Compositions and methods for immunotherapy

ABSTRACT

The present invention provides for methods and compositions for enhancing the immune response toward cancers and pathogens. It relates to immunoresponsive cells bearing antigen receptors, which can be chimeric antigen receptors (CARs), which express introduced ligands for immunomodulatory molecules. In particular embodiments, engineered immunoresponsive cells are antigen-directed and resist immunosuppression and/or have enhances immune-activating properties.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/148,687, filed Oct. 1, 2018, which is a divisional of U.S. patentapplication Ser. No. 14/835,264, filed Aug. 25, 2015, and is issued asU.S. Pat. No. 10,124,023, which is a continuation of InternationalPatent Application No. PCT/US2014/018667, filed Feb. 26, 2014 and claimspriority to U.S. Provisional Application No. 61/769,543, filed Feb. 26,2013, the contents of each of which are incorporated by reference intheir entirety, and to each of which priority is claimed.

GRANT INFORMATION

This invention was made with government support under Grant Nos.CA95152, CA138738, CA059350, and CA08748 from the National Institutes ofHealth. The government has certain rights in the invention.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted herewith via EFS on Aug. 12, 2021. Pursuant to 37 C.F.R. §1.52(e)(5), the Sequence Listing text file, identified as“0893330405SL.TXT,” is 74,704 bytes and was created on Aug. 12, 2021.The Sequence Listing, electronically filed herewith, does not extendbeyond the scope of the specification and thus does not contain newmatter.

INTRODUCTION

The present invention provides for methods and compositions forenhancing the immune response toward cancers and pathogens. It relatesto immunoresponsive cells bearing antigen receptors, which can bechimeric antigen receptors (CARs), that express introduced ligands forimmunomodulatory molecules. These engineered immunoresponsive cells areantigen-directed and resist immunosuppression and/or have enhancedimmune-activating properties.

BACKGROUND OF THE INVENTION

The majority of adult B-cell malignancies, including acute lymphoblasticleukemia (ALL), chronic lymphocytic leukemia, and non-Hodgkin'slymphoma, are incurable despite currently a vailable therapies. Adoptivetherapy with genetically engineered autologous T cells has shownevidence of therapeutic efficacy in melanoma and indolent B cellmalignancies. T cells may be modified to target tumor-associatedantigens through the introduction of genes encoding artificial T-cellreceptors, termed chimeric antigen receptors (CAR), specific to suchantigens. Immunotherapy is a targeted therapy that has the potential toprovide for the treatment of cancer.

However, malignant cells adapt to generate an immunosuppressivemicroenvironment to protect themselves from immune recognition andelimination. This “hostile” tumor microenvironment poses a challenge tomethods of treatment involving stimulation of an immune response, suchas targeted T cell therapies. Accordingly, novel therapeutic strategiesfor treating neoplasia are urgently required.

SUMMARY OF THE INVENTION

The present invention generally provides immunoresponsive cells (e.g., Tcells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTLs), andregulatory T cells), expressing an antigen binding receptor (e.g., CARor TCR) having immune cell activating activity and a single-chainvariable fragment (scFv) that binds an antigen having immunosuppressiveactivity (e.g., CD47, PD-1, CTLA-4, and ligands thereof), therebyreducing or eliminating the immunosuppressive activity of the antigen.

The invention further provides immunoresponsive cells (e.g., T cells,Natural Killer (NK) cells, cytotoxic T lymphocytes (CTLs), andregulatory T cells), expressing an antigen binding receptor (e.g., CARor TCR) having immune cell activating activity and a single-chainvariable fragment (scFv) that binds an antigen having immunostimulatoryor proinflammatory activity (e.g., CD28, OX-40, 4-1BB, CD40 and ligandsthereof), thereby enhancing the immunostimulatory activity of theantigen.

The invention further provides immunoresponsive cells (e.g., T cells,Natural Killer (NK) cells, cytotoxic T lymphocytes (CTLs), andregulatory T cells), expressing an antigen binding receptor (e.g., CARor TCR) having immune cell activating activity and CD40L, for example,exogenous CD40L (CD40L that has been introduced directly or indirectlyinto the cell (for example, via a vector of naked nucleic acidcomprising a nucleic acid sequence encoding CD40L), as compared toendogenous CD40L arising in the cell itself), thereby enhancing theimmunostimulatory activity of the antigen.

Accordingly, the invention provides methods of using suchimmunoresponsive cells for the treatment of neoplasia, infectiousdisease, and other pathologies.

In one aspect, the invention provides an isolated immunoresponsive cellhaving an antigen recognizing receptor that binds an antigen, where thebinding activates the immunoreponsive cell, and a soluble single-chainvariable fragment (scFv) that binds a polypeptide that hasimmunosuppressive activity or immunostimulatory activity.

In another aspect, the invention provides a method of treating orpreventing neoplasia in a subject, the method comprising administering,to the subject, an effective amount of an immunoresponsive cell havingan antigen recognizing receptor that binds a first antigen, where thebinding activates the immunoreponsive cell, and a soluble single-chainvariable fragment (scFv) that binds a polypeptide that hasimmunosuppressive activity or immunostimulatory activity, therebytreating or preventing neoplasia in the subject. In non-limitingembodiments, the antigen recognizing receptor is a CAR.

In another aspect, the invention provides a method of reducing tumorburden in a subject, the method involving administering, to the subject,an effective amount of an immunoresponsive cell having an antigenrecognizing receptor that binds a first antigen, where the bindingactivates the immunoreponsive cell, and a soluble single-chain variablefragment (scFv) that binds a polypeptide that has immunosuppressiveactivity or immunostimulatory activity, thereby inducing tumor celldeath in the subject. In still another aspect, the invention provides amethod of lengthening survival of a subject having neoplasia, the methodinvolving administering, to the subject, an effective amount of animmunoresponsive cell having an antigen recognizing receptor that bindsa first antigen, where the binding activates the immunoreponsive cell,and a soluble single-chain variable fragment (scFv) that binds apolypeptide that has immunosuppressive activity or immunostimulatoryactivity, thereby lengthening survival of the subject. In non-limitingembodiments, the antigen recognizing receptor is a CAR.

In various non-limiting embodiments, the invention provides a method ofincreasing immune-activating cytokine production in response to a cancercell in a subject, comprising administering, to the subject, animmunoresponsive cell having an antigen recognizing receptor that bindsan antigen of the cancer cell and further expressing exogenous CD40L. Inparticular non-limiting embodiments, the immune-activating cytokine isselected from the group consisting of. In a particular non-limitingembodiment, the immune-activating cytokine is IL-12. In non-limitingembodiments, the antigen recognizing receptor is a CAR.

In various non-limiting embodiments, the invention provides a method ofincreasing immune-activating cytokine production in response to apathogen in a subject, comprising administering, to the subject, animmunoresponsive cell having an antigen recognizing receptor that bindsan antigen of the pathogen and further expressing exogenous CD40L. Inparticular non-limiting embodiments, the immune-activating cytokine isselected from the group consisting of. In a particular non-limitingembodiment, the immune-activating cytokine is IL-12. In non-limitingembodiments, the antigen recognizing receptor is a CAR.

In various non-limiting embodiments, the invention provides a method ofincreasing a CD8⁺ cytotoxic T cell response to a cancer cell in asubject, comprising administering, to the subject, an immunoresponsivecell having an antigen recognizing receptor that binds an antigen of thecancer cell and further expressing exogenous CD40L. In non-limitingembodiments, the antigen recognizing receptor is a CAR.

In various non-limiting embodiments, the invention provides a method ofincreasing a CD8⁺ cytotoxic T cell response to a pathogen in a subject,comprising administering, to the subject, an immunoresponsive cellhaving an antigen recognizing receptor that binds an antigen of thepathogen and further expressing exogenous CD40L. In non-limitingembodiments, the antigen recognizing receptor is a CAR.

In various non-limiting embodiments, the invention provides a method ofpromoting dendritic cell maturation in a subject having a cancer,comprising administering, to the subject, an immunoresponsive cellhaving an antigen recognizing receptor that binds an antigen of a cellof the cancer and further expressing exogenous CD40L. In non-limitingembodiments, the antigen recognizing receptor is a CAR.

In various non-limiting embodiments, the invention provides a method ofpromoting dendritic cell maturation in a subject having a disease causedby a pathogen, comprising administering, to the subject, animmunoresponsive cell having an antigen recognizing receptor that bindsan antigen of the pathogen and further expressing exogenous CD40L. Innon-limiting embodiments, the antigen recognizing receptor is a CAR.

In still another aspect, the invention provides a method of treating orpreventing neoplasia in a subject, the method comprising administering,to the subject, an effective amount of an immunoresponsive cell havingan antigen recognizing receptor that binds a first antigen, where thebinding activates the immunoreponsive cell, and expressing exogenousCD40L, thereby treating or preventing a neoplasia in the subject. Innon-limiting embodiments, the antigen recognizing receptor is a CAR.

In another aspect, the invention provides a method of reducing tumorburden in a subject, the method involving administering, to the subject,an effective amount of an immunoresponsive cell having an antigenrecognizing receptor that binds a first antigen, where the bindingactivates the immunoreponsive cell, and expressing exogenous CD40L,thereby inducing tumor cell death in the subject. In still anotheraspect, the invention provides a method of lengthening survival of asubject having neoplasia, the method involving administering, to thesubject, an effective amount of an immunoresponsive cell having anantigen recognizing receptor that binds a first antigen, where thebinding activates the immunoreponsive cell, and expressing exogenousCD40L, thereby lengthening survival of the subject.

In yet another aspect, the invention provides a method of treating bloodcancer in a subject in need thereof, the method involving administeringto the subject a therapeutically effective amount of a T cell having anantigen recognizing receptor that binds CD19, where the bindingactivates the immunoreponsive cell, and a soluble single-chain variablefragment (scFv) that binds one or more of CD47, PD-1, CTLA-4, andligands thereof, thereby treating blood cancer in the subject. Innon-limiting embodiments, the antigen recognizing receptor is a CAR.

In yet another aspect, the invention provides a method of treating bloodcancer in a subject in need thereof, the method involving administeringto the subject a therapeutically effective amount of a T cell having anantigen recognizing receptor that binds CD19, where the bindingactivates the immunoreponsive cell, and expressing exogenous CD40L,thereby treating blood cancer in the subject. In non-limitingembodiments, the antigen recognizing receptor is a CAR.

In one aspect, the invention provides a method for producing anantigen-specific immunoresponsive cell, the method involving introducinginto the immunoresponsive cell a nucleic acid sequence that encodes asingle-chain variable fragment (scFv) that binds a polypeptide that hasimmunosuppressive activity or immunostimulatory activity, where theimmunoresponsive cell has an antigen recognizing receptor that binds anantigen. In non-limiting embodiments, the antigen recognizing receptoris a CAR.

In one aspect, the invention provides a method for producing anantigen-specific immunoresponsive cell, the method involving introducinginto the immunoresponsive cell a nucleic acid sequence that encodesCD40L, where the immunoresponsive cell has an antigen recognizingreceptor that binds an antigen. In non-limiting embodiments, the nucleicacid sequence that encodes CD40L is operably linked to a promoterelement constitutively or inducibly expressed in the immunoresponsivecell, optionally comprised in a vector. In non-limiting embodiments, theantigen recognizing receptor is a CAR. In non-limiting embodiments, theinvention provides for a nucleic acid comprising sequence encoding a CARand encoding CD40L, each optionally operably linked to a promoterelement constitutively or inducibly expressed in the immunoresponsivecell, and said nucleic acid may optionally be comprised in a vector. Inone aspect, the invention provides a vector having a nucleic acidsequence encoding an antigen recognizing receptor that binds an antigen,and a nucleic acid sequence encoding a soluble single-chain variablefragment (scFv) that binds a polypeptide having immunosuppressiveactivity or immunostimulatory activity. In non-limiting embodiments, theantigen recognizing receptor is a CAR.

In one aspect, the invention provides a vector having a nucleic acidsequence encoding an antigen recognizing receptor that binds an antigen,and a nucleic acid sequence encoding CD40L. In non-limiting embodiments,the antigen recognizing receptor is a CAR. In one specific non-limitingembodiment, the invention provides for a retroviral vector containing ananti-CD19 CAR (1928z)- and a CD40L-encoding nucleic acid.

In a related aspect, the invention provides a pharmaceutical compositioncontaining an effective amount of an immunoresponsive cell of any aspectof the invention delineated herein in a pharmaceutically acceptableexcipient. In another related aspect, the invention provides apharmaceutical composition for the treatment of a neoplasia containingan effective amount of a tumor antigen-specific T cell of any aspect ofthe invention delineated herein in a pharmaceutically acceptableexcipient.

In an additional aspect, the invention provides a kit for treatment of aneoplasia, pathogen infection, an autoimmune disorder, or an allogeneictransplant, the kit containing an immunoresponsive cell having anantigen recognizing receptor that binds an antigen and activates theimmunoreponsive cell, and a soluble single-chain variable fragment(scFv) that binds a polypeptide that has immunosuppressive activity orimmunostimulatory activity. In non-limiting embodiments, the antigenrecognizing receptor is a CAR. In particular embodiments, the kitfurther contains written instructions for using the cell for thetreatment of a subject having a neoplasia, a pathogen infection, anautoimmune disorder, or an allogeneic transplant.

In an additional aspect, the invention provides a kit for treatment of aneoplasia, pathogen infection, an autoimmune disorder, or an allogeneictransplant, the kit containing an immunoresponsive cell having anantigen recognizing receptor that binds an antigen and activates theimmunoreponsive cell, and expressing exogenous CD40L. In non-limitingembodiments, the antigen recognizing receptor is a CAR. In particularembodiments, the kit further contains written instructions for using thecell for the treatment of a subject having a neoplasia, a pathogeninfection, an autoimmune disorder, or an allogeneic transplant.

In an additional aspect, the invention provides a kit for treatment of aneoplasia, pathogen infection, an autoimmune disorder, or an allogeneictransplant, the kit comprising a nucleic acid encoding a CAR whichrecognizes an antigen of the neoplasia, pathogen, autoimmune disorder,or transplant to be treated, and a nucleic acid encoding a solublesingle-chain variable fragment (scFv) that binds a polypeptide that hasimmunosuppressive activity or immunostimulatory activity. Optionally oneor both nucleic acids may be comprised in a vector, which may be thesame vector (bicistronic) or separate vectors. The nucleic acid encodingthe CAR and/or the nucleic acid encoding the scFv may each be operablylinked to a promoter which may be the same or different promoters. Inparticular embodiments, the kit further contains written instructionsfor using the cell for the treatment of a subject having a neoplasia, apathogen infection, an autoimmune disorder, or an allogeneic transplant.

In an additional aspect, the invention provides a kit for treatment of acancer, the kit comprising a nucleic acid encoding a CAR whichrecognizes an antigen of the cancer, and a nucleic acid encoding asoluble single-chain variable fragment (scFv) that binds a polypeptidethat has immunosuppressive activity or immunostimulatory activity.Optionally one or both nucleic acids may be comprised in a vector, whichmay be the same vector (bicistronic) or separate vectors. The nucleicacid encoding the CAR and/or the nucleic acid encoding the scFv may eachbe operably linked to a promoter which may be the same or differentpromoters. In particular embodiments, the kit further contains writteninstructions for using the cell for the treatment of a subject having acancer.

In an additional aspect, the invention provides a kit for treatment of acancer or pathogen-mediated disorder, the kit comprising a nucleic acidencoding a CAR which recognizes an antigen of the cancer or pathogen,and a nucleic acid encoding a soluble single-chain variable fragment(scFv) that binds a polypeptide that has immunosuppressive activity orimmunostimulatory activity. Optionally one or both nucleic acids may becomprised in a vector, which may be the same vector (bicistronic) orseparate vectors. The nucleic acid encoding the CAR and/or the nucleicacid encoding the scFv may each be operably linked to a promoter whichmay be the same or different promoters. In particular embodiments, thekit further contains written instructions for using the cell for thetreatment of a subject having a cancer or disorder. In an additionalaspect, the invention provides a kit for treatment of a cancer orpathogen-mediated disorder, the kit comprising a nucleic acid encoding aCAR which recognizes an antigen of the cancer or pathogen, and a nucleicacid encoding CD40L. Optionally one or both nucleic acids may becomprised in a vector, which may be the same vector (bicistronic) orseparate vectors. The nucleic acid encoding the CAR and/or the nucleicacid encoding CD40L may each be operably linked to a promoter which maybe the same or different promoters. In particular embodiments, the kitfurther contains written instructions for using the cell for thetreatment of a subject having a cancer or disorder.

In various embodiments of any of the aspects delineated herein, the cellis selected from the group consisting of a T cell, a Natural Killer (NK)cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a humanembryonic stem cell, and a pluripotent stem cell from which lymphoidcells may be differentiated. In various embodiments of any of theaspects delineated herein, the immunoresponsive cell is autologous.

In various embodiments of any of the aspects delineated herein, theantigen recognizing receptor is a T cell receptor (TCR) or chimericantigen receptor (CAR). In various embodiments of any of the aspectsdelineated herein, the antigen recognizing receptor is exogenous orendogenous. In various embodiments of any of the aspects delineatedherein, the antigen recognizing receptor is recombinantly expressed. Invarious embodiments of any of the aspects delineated herein, the antigenrecognizing receptor is expressed from a vector. In various embodimentsof any of the aspects delineated herein, the intracellular signalingdomain of the antigen recognizing receptor is the CDC-chain, CD97,CD11a-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-IBB, CD28 signalingdomain, a portion thereof, or combinations thereof. In non-limitingembodiments, the antigen recognizing receptor is a CAR comprising atleast a portion of CD28, 4-IBB, and/or CD3ζ-chain (see, e.g., Zhong etal., 2010, Molecular Ther. 18(2):413-420), together with an antigenbinding portion. In non-limiting embodiments, the antigen recognizingreceptor is a CAR described in Kohn et al., 2011, Molecular Ther.19(3):432-438), optionally where the antigen binding portion issubstituted with amino acid sequence that binds to another tumor orpathogen antigen. In various embodiments, the cell expresses arecombinant or an endogenous antigen receptor that is 1928z or 4H1128z.

In various embodiments of any of the aspects delineated herein, theantigen is a tumor or pathogen antigen. In various embodiments of any ofthe aspects delineated herein, the tumor antigen is one or more of CD19,MUC16, MUC1, CA1X, CEA, CDS, CD7, CD10, CD20, CD22, CD30, CD33, CD34,CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, a cytomegalovirus(CMV) infected cell antigen, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP,Fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT,IL-13R-a2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1,Mesothelin, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4), PSCA,PSMA, ROR1, TAG-72, VEGF-R2, or WT-1. In particular embodiments, theantigen is CD19 or MUC16. Amino acid sequences that specifically bind tosaid antigens are known in the art or may be prepared using methodsknown in the art; examples include immunoglobulins, variable regions ofimmunoglobulins (e.g. variable fragment (“Fv”) or bivalent variablefragment (“Fab”)), single chain antibodies, etc.

In various embodiments of any of the aspects delineated herein, thesoluble scFv is secreted. In various embodiments of any of the aspectsdelineated herein, the scFv is expressed from a vector. In variousembodiments of any of the aspects delineated herein, theimmunosuppressive polypeptide is one or more of CD47, PD-1, CTLA-4, andligands thereof. In various embodiments of any of the aspects delineatedherein, the immunostimulatory polypeptide is one or more of CD28, OX-40,4-1BB, and ligands thereof. In various embodiments of any of the aspectsdelineated herein, the soluble scFv enhances an immune response of theimmunoreponsive cell.

In various embodiments of any of the aspects delineated herein, theimmunoresponsive cell secretes a cytokine. In various embodiments of anyof the aspects delineated herein, the cytokine is expressed from avector. In various embodiments of any of the aspects delineated herein,the pharmaceutical composition containing an immunoresponsive cell ofthe invention contains a cytokine. In various embodiments of any of theaspects delineated herein, an immunoresponsive cell of the invention isadministered with a cytokine. In various embodiments of any of theaspects delineated herein, the cytokine is one or more of IL-2, IL-3,IL-6, IL-11, IL7, IL12, IL15, IL21, granulocyte macrophage colonystimulating factor, alpha, beta or gamma interferon and erythropoietin.

In various embodiments of any of the aspects delineated herein, themethod reduces the number of tumor cells, reduces tumor size, eradicatesthe tumor in the subject, reduces the tumor burden in the subject,and/or eradicates the tumor burden in the subject.

Illustrative neoplasms for which the invention can be used include, butare not limited to leukemias (e.g., acute leukemia, acute lymphocyticleukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acute monocyticleukemia, acute erythroleukemia, chronic leukemia, chronic myelocyticleukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma(Hodgkin's disease, non-Hodgkin's disease), Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors such assarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).

In various non-limiting embodiments of any of the aspects delineatedherein, the neoplasia is one or more of blood cancer, B cell leukemia,multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocyticleukemia, non-Hodgkin's lymphoma, and ovarian cancer. In certainembodiments, the blood cancer is one or more of B cell leukemia,multiple myeloma, acute lymphoblastic leukemia (ALL), chroniclymphocytic leukemia, and non-Hodgkin's lymphoma. In particularembodiments, the neoplasia is B cell leukemia, the antigen is CD19, andthe polypeptide that has immunosuppressive activity is one or more ofCD47, PD-1, CTLA-4, and ligands thereof. In particular embodiments, theneoplasia is multiple myeloma, the antigen is CD19, and the polypeptidethat has immunosuppressive activity is one or more of CD47, PD-1,CTLA-4, and ligands thereof. In particular embodiments, the neoplasia isacute lymphoblastic leukemia (ALL), the antigen is CD19, and thepolypeptide that has immunosuppressive activity is one or more of CD47,PD-1, CTLA-4, and ligands thereof. In particular embodiments, theneoplasia is chronic lymphocytic leukemia, the antigen is CD19, and thepolypeptide that has immunosuppressive activity is one or more of CD47,PD-1, CTLA-4, and ligands thereof. In particular embodiments, theneoplasia is non-Hodgkin's lymphoma, the antigen is CD19, and thepolypeptide that has immunosuppressive activity is one or more of CD47,PD-1, CTLA-4, and ligands thereof. In particular embodiments, theneoplasia is ovarian cancer, the antigen is MUC16, and the polypeptidethat has immunosuppressive activity is one or more of CD47, PD-1,CTLA-4, and ligands thereof.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “activates an immunoresponsive cell” is meant induction of signaltransduction or changes in protein expression in the cell resulting ininitiation of an immune response. For example, when CD3 Chains clusterin response to ligand binding and immunoreceptor tyrosine-basedinhibition motifs (ITAMs) a signal transduction cascade is produced. Incertain embodiments, when an endogenous TCR or an exogenous CAR bindsantigen, a formation of an immunological synapse occurs that includesclustering of many molecules near the bound receptor (e.g. CD4 or CD8,CD3γ/δ/ε/ζ, etc.) This clustering of membrane bound signaling moleculesallows for ITAM motifs contained within the CD3 chains to becomephosphorylated. This phosphorylation in turn initiates a T cellactivation pathway ultimately activating transcription factors, such asNF-κB and AP-1. These transcription factors induce global geneexpression of the T cell to increase IL-2 production for proliferationand expression of master regulator T cell proteins in order to initiatea T cell mediated immune response. By “stimulates an immunoresponsivecell” is meant a signal that results in a robust and sustained immuneresponse. In various embodiments, this occurs after immune cell (e.g.,T-cell) activation or concomitantly mediated through receptorsincluding, but not limited to, CD28, CD137 (4-1BB), OX40, CD40 and ICOS.Without being bound to a particular theory, receiving multiplestimulatory signals is important to mount a robust and long-term T cellmediated immune response. Without receiving these stimulatory signals, Tcells quickly become inhibited and unresponsive to antigen. While theeffects of these co-stimulatory signals vary and remain partiallyunderstood, they generally result in increasing gene expression in orderto generate long lived, proliferative, and anti-apoptotic T cells thatrobustly respond to antigen for complete and sustained eradication.

The term “antigen recognizing receptor” as used herein refers to areceptor that is capable of activating an immune cell (e.g., a T-cell)in response to antigen binding. Exemplary antigen recognizing receptorsmay be native or endogenous T cell receptors or chimeric antigenreceptors in which a tumor antigen-binding domain is fused to anintracellular signaling domain capable of activating an immune cell(e.g., a T-cell).

As used herein, the term “antibody” means not only intact antibodymolecules, but also fragments of antibody molecules that retainimmunogen-binding ability. Such fragments are also well known in the artand are regularly employed both in vitro and in vivo. Accordingly, asused herein, the term “antibody” means not only intact immunoglobulinmolecules but also the well-known active fragments F(ab′)₂, and Fab.F(ab′)₂, and Fab fragments that lack the Fe fragment of intact antibody,clear more rapidly from the circulation, and may have less non-specifictissue binding of an intact antibody (Wahl et al., J. Nucl. Med.24:316-325 (1983). The antibodies of the invention comprise whole nativeantibodies, bispecific antibodies; chimeric antibodies; Fab, Fab′,single chain V region fragments (scFv), fusion polypeptides, andunconventional antibodies.

As used herein, the term “single-chain variable fragment” or “scFv” is afusion protein of the variable regions of the heavy (VH) and lightchains (VL) of an immunoglobulin covalently linked to form a VH::VLheterodimer. The heavy (VH) and light chains (VL) are either joineddirectly or joined by a peptide-encoding linker (e.g., 10, 15, 20, 25amino acids), which connects theN-terminus of the VH with the C-terminusof the VL, or the C-terminus of the VH with theN-terminus of the VL. Thelinker is usually rich in glycine for flexibility, as well as serine orthreonine for solubility. Despite removal of the constant regions andthe introduction of a linker, scFv proteins retain the specificity ofthe original immunoglobulin. Single chain Fv polypeptide antibodies canbe expressed from a nucleic acid including VH- and VL-encoding sequencesas described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883,1988). See, also, U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,956,778; andU.S. Patent Publication Nos. 20050196754 and 20050196754. AntagonisticscFvs having inhibitory activity have been described (see, e.g., Zhao etal., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J CachexiaSarcopenia Muscle 2012 Aug. 12; Shieh et al., J Imunol2009183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63;Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al.,Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 19952(10:31-40). Agonistic scFvs having stimulatory activity have beendescribed (see, e.g., Peter et al., J Biol Chern 2003 25278(38):36740-7;Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit RevImmunol1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 20031638(3):257-66).

By “affinity” is meant a measure of binding strength. Without beingbound to theory, affinity depends on the closeness of stereochemical fitbetween antibody combining sites and antigen determinants, on the sizeof the area of contact between them, and on the distribution of chargedand hydrophobic groups. Affinity also includes the term “avidity,” whichrefers to the strength of the antigen-antibody bond after formation ofreversible complexes. Methods for calculating the affinity of anantibody for an antigen are known in the art, including use of bindingexperiments to calculate affinity. Antibody activity in functionalassays (e.g., flow cytometry assay) is also reflective of antibodyaffinity. Antibodies and affinities can be phenotypically characterizedand compared using functional assays (e.g., flow cytometry assay).

The term “chimeric antigen receptor” or “CAR” as used herein refers toan antigen-binding domain that is fused to an intracellular signalingdomain capable of activating or stimulating an immune cell. Mostcommonly, the CAR's extracellular binding domain is composed of a singlechain variable fragment (scFv) derived from fusing the variable heavyand light regions of a murine or humanized monoclonal antibody.Alternatively, scFvs may be used that are derived from Fab's (instead offrom an antibody, e.g., obtained from Fab libraries). In variousembodiments, this scFv is fused to a transmembrane domain and then to anintracellular signaling domain. “First-generation” CARs include thosethat solely provide CD3ζ signals upon antigen binding,“Second-generation” CARs include those that provide both costimulation(e.g. CD28 or CD137) and activation (CD3ζ). “Third-generation” CARsinclude those that provide multiple costimulation (e.g. CD28 and CD137)and activation (CD3ζ). In various embodiments, the CAR is selected tohave high affinity or avidity for the antigen.

The term “immunosuppressive activity” is meant induction of signaltransduction or changes in protein expression in a cell (e.g., anactivated immunoresponsive cell) resulting in a decrease in an immuneresponse. Polypeptides known to suppress or decrease an immune responsevia their binding include CD47, PD-1, CTLA-4, and their correspondingligands, including SIRPa, PD-L1, PD-L2, B7-1, and B7-2. Suchpolypeptides are present in the tumor microenvironment and inhibitimmune responses to neoplastic cells. In various embodiments,inhibiting, blocking, or antagonizing the interaction ofimmunosuppressive polypeptides and/or their ligands enhances the immuneresponse of the immunoresponsive cell.

The term “immunostimulatory activity” is meant induction of signaltransduction or changes in protein expression in a cell (e.g., anactivated immunoresponsive cell) resulting in an increase in an immuneresponse. Immunostimulatory activity may include pro-inflammatoryactivity. Polypeptides known to stimulate or increase an immune responsevia their binding include CD28, OX-40, 4-1BB, and their correspondingligands, including B7-1, B7-2, OX-40L, and 4-1BBL. Such polypeptides arepresent in the tumor microenvironment and activate immune responses toneoplastic cells. In various embodiments, promoting, stimulating, oragonizing pro-inflammatory polypeptides and/or their ligands enhancesthe immune response of the immunoreponsive cell.

By “CD3ζ polypeptide” is meant a protein having at least 85, 90, 95, 96,97, 98, 99 or 100% identity to NCBI Reference No: NP_932170 or afragment thereof that has activating or stimulatory activity. Anexemplary CD3ζ; is provided below [SEQ ID NO:1].

  1 mkwkalftaa ilqaqlpite aqsfglldpk lcylldgilf iygviltalf lrvkfsrsad 61 apayqqgqnq lynelnlgrr eeydvldkrr grdpemggkp qrrknpqegl ynelqkdkma121 eayseigmkg errrgkghdg lyqglstatk dtydalhmqa lppr

By “CD3ζ nucleic acid molecule” is meant a polynucleotide encoding aCD3ζ polypeptide.

By “CD28 polypeptide” is meant a protein having at least 85, 90, 95, 96,97, 98, 99 or 100% identity to NCBI Reference No: NP_006130 or afragment thereof that has stimulatory activity. An exemplary CD28 isprovided below [SEQ ID NO:2].

  1 mlrlllalnl fpsiqvtgnk ilvkqspmlv aydnavnlsc kysynlfsre fraslhkgld 61 savevcvvyg nysqqlqvys ktgfncdgkl gnesvtfylq nlyvnqtdiy fckievmypp121 pyldneksng tiihvkgkhl cpsplfpgps kpfwvlvvvg gvlacysllv tvafiifwvr181 skrsrllhsd ymnmtprrpg ptrkhyqpya pprdfaayrs

By “CD28 nucleic acid molecule” is meant a polynucleotide encoding aCD28 polypeptide.

By “CD40L polypeptide” is meant a protein having at least 85, 90, 95,96, 97, 98, 99 or 100% identity to NCBI Reference Sequence: NP_000065,GenBank Reference No. GenBank: AAH74950.1 or a fragment thereof that isa CD40 ligand, or a protein encoded by a nucleic acid PCR amplified fromisolated healthy donor PBMCs using the following primers (1)5′-CACGTGCATGATCGAAACATACAACCAAACTTCTCCCCGATCTGC-′3 [SEQ ID NO:3] and(2) 5′-CTCGAGGGATCCTCAGAGTTTGAGTAAGCCAAAGGA-3′ [SEQ ID NO:4](FIG. 22A).

By “CD40L nucleic acid molecule” is meant a polynucleotide encoding aCD40L polypeptide.

By “4-1BB polypeptide” is meant a protein having at least 85, 90, 95,96, 97, 98, 2599 or 100% identity to NCBI Reference No: P41273 orNP_001552 or a fragment thereof that that acts as a tumor necrosisfactor (TNF) ligand. An exemplary 4-1BB is provided below [SEQ ID NO:5].

1 mgnscyniva tlllvlnfer trslqdpcsn cpagtfcdnn rnqicspcpp nsfssaggqr 61tcdicrqckg vfrtrkecss tsnaecdctp gfhclgagcs mceqdckqgq eltkkgckdc 121cfgtfndqkr gicrpwtncs ldgksvlvng tkerdvvcgp spadlspgas svtppapare 181pghspqiisf flaltstall fllffltlrf svvkrgrkkl lyifkqpfmr pvqttgeedg 241cscrfpeeee ggcel

By “4-1BBL nucleic acid molecule” is meant a polynucleotide encoding a4-1BBL polypeptide.

By “OX40L polypeptide” is meant a protein having at least 85, 90, 95,96, 97, 98, 99 or 100% identity to NCBI Reference No: BAB18304 orNP_003317 or a fragment thereof that is a tumor necrosis factor (TNF)ligand [SEQ ID NO:6].

  1 mervqpleen vgnaarprfe rnklllvasv    igglglllcf tyiclhfsal qvshrypriq 61 sikvqfteyk kekgfiltsq kedeimkvqn    nsviincdgf ylislkgyfs qevnislhyq121 kdeeplfqlk kvrsvnslmv asltykdkvy    lnvttdntsl ddfhvnggel ilihqnpgef 181 cvl

By “OX40L nucleic acid molecule” is meant a polynucleotide encoding aOX40L polypeptide.

By “1928z” is meant a protein having at least 85, 90, 95, 96, 97, 98, 99or 100% identity to the sequence provided below, which includes a CDSleader sequence at amino acids 1-18, and is able to bind CD19 [SEQ IDNO:7].

MALPVTALLLPLALLLHAEVKLQQSGA ELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGK FKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTT VTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVA WYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFC QQYNRYPYTSGGGTKLEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP LFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRSRVKFSRSAEPPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPRX

An exemplary nucleic acid sequence encoding a 1928z polypeptide,including a CDS leader sequence, is provided below [SEQ ID NO:8].

ccatggctctcccagtgactgccctac tgcttcccctagcgcttctcctgcatgcagaggtgaagctgcagcagtctgggg ctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggct atgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtc ttgagtggattggacagatttatcctggagatggtgatactaactacaatggaa agttcaagggtcaagccacactgactgcagacaaatcctccagcacagcctaca tgcagctcagcggcctaacatctgaggactctgcggtctatttctgtgcaagaa agaccattagttcggtagtagatttctactttgactactggggccaagggacca cggtcaccgtctcctcaggtggaggtggatcaggtggaggtggatctggtggag gtggatctgacattgagctcacccagtctccaaaattcatgtccacatcagtag gagacagggtcagcgtcacctgcaaggccagtcagaatgtgggtactaatgtag cctggtatcaacagaaaccaggacaatctcctaaaccactgatttactcggcaa cctaccggaacagtggagtccctgatcgcttcacaggcagtggatctgggacag atttcactctcaccatcactaacgtgcagtctaaagacttggcagactatttct gtcaacaatataacaggtatccgtacacgtccggaggggggaccaagctggaga tcaaacgggcggccgcaattgaagttatgtatcctcctccttacctagacaatg agaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtc ccctatttcccggaccttctaagcccttttgggtgctggtggtggttggtggag tcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtga ggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgcc gccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcg cagcctatcgctccagagtgaagttcagcaggagcgcagagccccccgcgtacc agcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagt acgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccga gaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatgg cggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggc acgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgccc ttcacatgcaggccctgccccctcgcg

By “4H1128z” is meant a protein having at least 85, 90, 95, 96, 97, 98,99 or 100% identity to the sequence provided below, which includes a CDSleader sequence at amino acids 1-18, and is able to bind MUC [SEQ IDNO:9].

MALPVTALLLPLALLLHAEVKLQESGG GFVKPGGSLKVSCAASGFTFSSYAMSWVRLSPEMRLEWVATISSAGGYIFYSDS VQGRFTISRDNAKNTLHLQMGSLRSGDTAMYYCARQGFGNYGDYYAMDYWGQGT TVTVSSGGGGSGGGGSGGGGSDIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSR TRKNQLAWYQQKPGQSPELLIYWASTRQSGVPDRFTGSGSGTDFTLTISSVQAE DLAVYYCQQSYNLLTFGPGTKLEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGK HLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM NMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSAEPPAYQQGQNQLYNELNL GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR

An exemplary nucleic acid sequence encoding a 4H1128z polypeptide,including a Kappa leader sequence, is provided below [SEQ ID NO:10].

ccatggctctcccagtgactgccctac tgcttcccctagcgcttctcctgcatgcagaggtgaagctgcaggagtcagggg gaggcttcgtgaagcctggagggtccctcaaagtctcctgtgcagcctctggat tcactttcagtagctatgccatgtcctgggttcgcctgagtccggagatgaggc tggagtgggtcgcaaccattagcagtgctggtggttacatcttctattctgaca gtgtgcagggacgattcaccatttccagagacaatgccaagaacaccctgcacc tgcaaatgggcagtctgaggtctggggacacggccatgtattactgtgcaaggc agggatttggtaactacggtgattactatgctatggactactggggccaaggga ccacggtcaccgtctcctcaggtggaggtggatcaggtggaggtggatctggtg gaggtggatctgacattgagctcacccagtctccatcctccctggctgtgtcag caggagagaaggtcactatgagctgcaaatccagtcagagtctgctcaacagta gaacccgaaagaaccagttggcttggtaccagcaaaaaccaggacagtctcctg aactgctgatctactgggcatccactaggcaatctggagtccctgatcgcttca caggcagtggatctgggacagatttcactctcaccatcagcagtgtgcaggctg aagacctggcagtttattactgccagcaatcttataatctactcacgttcggtc ctgggaccaagctggagatcaaacgggcggccgcaattgaagttatgtatcctc ctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaaggga aacacctttgtccaagtcccctatttcccggaccttctaagccattttgggtgc tggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcct ttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactaca tgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatg ccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcg cagagccccccgcgtaccagcagggccagaaccagctctataacgagctcaatc taggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctg agatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaac tgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagc gccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccacca aggacacctacgacgcccttcacatgcaggccctgccccctcgc

By “B6H12.2 scFv” is meant a protein having at least 85, 90, 95, 96, 97,98, 99 or 100% identity to the sequence provided below and is able tobind CD47 [SEQ ID NO:11].

EVQLVESGGDLVKPGGSLKLSCAAS GFTFSGYGMSWVRQTPDKRLEWVATITSGGTYTYYPDSVKGRFTISRDNA KNTLYLQIDSLKSEDTAIYFCARSLAGNAMDYWGQGTSVTVSSGGGGSGG GGSGGGGSDIVMTQSPATLSVTPGDRVSLSCRASQTISDYLHWYQQKSHE SPRLLIKFASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGH GFPRTFGGGTKLEIKEQKLISEEDL

By “5C4 scFv” is meant a protein having at least 85, 90, 95, 96, 97, 98,99 or 100% identity to the sequence provided below and is able to bindhuman PD-1 [SEQ ID NO:12].

QVQLVESGGGVVQPGRSLRLDCKAS GITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNS KNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSGGGGSGGGGSGG GGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRT FGQGTKVEIK

By “J43 scFv” is meant a protein having at least 85, 90, 95, 96, 97, 98,99 or 100% identity to the sequence provided below, which includes aKappa leader sequence at amino acids 1-21, and is able to bind humanPD-1 [SEQ ID NO:13].

METDTLLLWVLLLWVPGSTGDMGLGLQ WVFFVALLKGVHCEVRLLESGGGLVKPEGSLKLSCVASGFTFSDYFMSWVRQAP GKGLEWVAHIYTKSYNYATYYSGSVKGRFTISRDDSRSMVYLQMNNLRTEDTAT YYCTRDGSGYPSLDFWGQGTQVTVSSATTTAPSVYPLAPACDSTTKSGGGGSGG GGSGGGGSYELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVI YDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDEGDYYCFSGYVDSDSKLYVF GSGTQLTVLGGPKSSPKVTVFPPSPEELRINKATLVCLVNDFYPGSATVTWKAN GATINDGVKITKPSKQGQNYMTSSYLSLTADQWKSHNRVSCQVTHEGETVEKSL SPAECLEQKLISEEDL*

An exemplary nucleic acid sequence encoding a J43 scFv polypeptide,including a Kappa leader sequence, is provided below [SEQ ID NO:14].

ccATGGAGACAGACACACTCCTGCTAT GGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACatgggattgggactgc agtgggttttctttgttgctcttttaaaaggtgtccactgtgaggtgcggcttc tggagtctggtggaggattagtgaagcctgaggggtcactgaaactctcctgtg tggcctctggattcaccttcagtgactatttcatgagctgggtccgccaggctc cagggaaggggctggagtgggttgctcacatatacacgaaaagttataattatg caacttattactcgggttcggtgaaaggcagattcaccatctccagagatgatt cccgaagcatggtctacctgcaaatgaacaacctgagaactgaggacacggcca cttattactgtacaagagatggaagcggatatccctctctggatttctggggtc aagggacccaagtcactgtctcctcagccacaacaacagccccatctgtctatc ccttggcccctgcctgtgacagcacaaccaaatcgggtggaggtggatcaggtg gaggtggatctggtggaggtggatctTatgagctgactcagccaccttcagcat cagtcaatgtaggagagactgtcaaaatcacctgctctggggaccaattgccga aatattttgcagattggtttcatcaaaggtcagaccagaccattttgcaagtga tatatgatgataataagcgcccctcggggatccctgaaagaatctctgggtcca gctcagggacaacagccaccttgaccatcagagatgtccgggctgaggatgaag gtgactattactgtttctcaggatatgttgatagtgatagcaaattgtatgttt ttggcagcggaacccagctcaccgtcctaggtggacccaagtcttctcccaaag tcacagtgtttccaccttcacctgaggagctccggacaaacaaagccacactgg tgtgtctggttaatgacttctacccgggttctgcaacagtgacctggaaggcaa atggagcaactatcaatgatggggtgaagactacaaagccttccaaacagggcc aaaactacatgaccagcagctacctaagtttgacagcagaccagtggaaatctc acaacagggtttcctgccaagttacccatgaaggggaaactgtggagaagagtt tgtcccctgcagaatgtctcgaacaaaaactcatatcagaagaggatatgTAAc tcgag

Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Rogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e-3 and e-100 indicating a closely related sequence.

By “analog” is meant a structurally related polypeptide or nucleic acidmolecule having the function of a reference polypeptide or nucleic acidmolecule.

The term “ligand” as used herein refers to a molecule that binds to areceptor. In particular, the ligand binds a receptor on another cell,allowing for cell-to-cell recognition and/or interaction.

The term “constitutive expression” as used herein refers to expressionunder all physiological conditions.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include neoplasia or pathogen infection of cell.

By “effective amount” is meant an amount sufficient to have atherapeutic effect. In one embodiment, an “effective amount” is anamount sufficient to arrest, ameliorate, or inhibit the continuedproliferation, growth, or metastasis (e.g., invasion, or migration) of aneoplasia.

By “endogenous” is meant a nucleic acid molecule or polypeptide that isnormally expressed in a cell or tissue.

By “enforcing tolerance” is meant preventing the activity ofself-reactive cells or immunoresponsive cells that target transplantedorgans or tissues.

By “exogenous” is meant a nucleic acid molecule or polypeptide that isnot endogenously present in the cell, or not present at a levelsufficient to achieve the functional effects obtained whenover-expressed. The term “exogenous” would therefore encompass anyrecombinant nucleic acid molecule or polypeptide expressed in a cell,such as foreign, heterologous, and over-expressed nucleic acid moleculesand polypeptides.

By a “heterologous nucleic acid molecule or polypeptide” is meant anucleic acid molecule (e.g., a cDNA, DNA or RNA molecule) or polypeptidethat is not normally present in a cell or sample obtained from a cell.This nucleic acid may be from another organism, or it may be, forexample, an mRNA molecule that is not normally expressed in a cell orsample.

By “immunoresponsive cell” is meant a cell that functions in an immuneresponse or a progenitor, or progeny thereof.

By “increase” is meant to alter positively by at least 5%. An alterationmay be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.

By “isolated cell” is meant a cell that is separated from the molecularand/or cellular components that naturally accompany the cell.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. A “purified” or“biologically pure” protein is sufficiently free of other materials suchthat any impurities do not materially affect the biological propertiesof the protein or cause other adverse consequences. That is, a nucleicacid or peptide of this invention is purified if it is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Purity and homogeneity aretypically determined using analytical chemistry techniques, for example,polyacrylamide gel electrophoresis or high performance liquidchromatography. The term “purified” can denote that a nucleic acid orprotein gives rise to essentially one band in an electrophoretic gel.For a protein that can be subjected to modifications, for example,phosphorylation or glycosylation, different modifications may give riseto different isolated proteins, which can be separately purified.

The term “tumor antigen-binding domain” as used herein refers to adomain capable of specifically binding a particular antigenicdeterminant or set of antigenic determinants present on a tumor.

The term “obtaining” as in “obtaining the agent” is intended to includepurchasing, synthesizing or otherwise acquiring the agent (or indicatedsubstance or material).

“Linker”, as used herein, shall mean a functional group (e.g., chemicalor polypeptide) that covalently attaches two or more polypeptides ornucleic acids so that they are connected to one another. As used herein,a “peptide linker” refers to one or more amino acids used to couple twoproteins together (e.g., to couple V_(H) and V_(L) domains). Anexemplary linker sequence used in the invention is GGGGSGGGGSGGGGS (SEQID NO: 51).

By “modulate” is meant positively or negatively alter. Exemplarymodulations include a 1%, 2%, 5%, 10%, 25%, 50%, 75%, or 100% change.

By “neoplasia” is meant a disease characterized by the pathologicalproliferation of a cell or tissue and its subsequent migration to orinvasion of other tissues or organs. Neoplasia growth is typicallyuncontrolled and progressive, and occurs under conditions that would notelicit, or would cause cessation of, multiplication of normal cells.Neoplasias can affect a variety of cell types, tissues, or organs,including but not limited to an organ selected from the group consistingof bladder, bone, brain, breast, cartilage, glia, esophagus, fallopiantube, gallbladder, heart, intestines, kidney, liver, lung, lymph node,nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin,spinal cord, spleen, stomach, testes, thymus, thyroid, trachea,urogenital tract, ureter, urethra, uterus, and vagina, or a tissue orcell type thereof. Neoplasias include cancers, such as sarcomas,carcinomas, or plasmacytomas (malignant tumor of the plasma cells).

By “pathogen” is meant a virus, bacteria, fungi, parasite or protozoacapable of causing disease.

Exemplary viruses include, but are not limited to, Retroviridae (e.g.human immunodeficiency viruses, such as HIV-1 (also referred to asHDTV-III, LAVE or HTLV-III/LAV, or HIV-III; and other isolates, such asHIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae(e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g.dengue viruses, encephalitis viruses, yellow fever viruses);Coronoviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicularstomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses);Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus,respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses);Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses andNaira viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae(e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae;Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (mostadenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2,varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae(variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g.African swine fever virus); and unclassified viruses (e.g. the agent ofdelta hepatitis (thought to be a defective satellite of hepatitis Bvirus), the agents of non-A, non-B hepatitis (class 1=internallytransmitted; class 2=parenterally transmitted (i.e. Hepatitis C);Norwalk and related viruses, and astroviruses).

Exemplary bacteria include, but are not limited to, Pasteurella,Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, andSalmonella species. Specific examples of infectious bacteria include butare not limited to, Helicobacter pyloris, Borelia burgdorferi,Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, Mavium, M intracellulare, M kansaii, M gordonae), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Haemophilus influenzae, Bacillus antracis, Corynebacteriumdiphtherias, corynebacterium sp., Erysipelothrix rhusiopathiae,Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes,Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp.,Fusobacterium nucleatum, Streptobacillus moniliformis, Treponemapallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomycesisraelli.

By “receptor” is meant a polypeptide, or portion thereof, present on acell membrane that selectively binds one or more ligand.

By “reduce” is meant to alter negatively by at least 5%. An alterationmay be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.

By “recognize” is meant selectively binds a target. AT cell thatrecognizes a virus typically expresses a receptor that binds an antigenexpressed by the virus.

By “reference” or “control” is meant a standard of comparison. Forexample, the level of scFv-antigen binding by a cell expressing a CARand an scFv may be compared to the level of scFv-antigen binding in acorresponding cell expressing CAR alone.

By “secreted” is meant a polypeptide that is released from a cell viathe secretory pathway through the endoplasmic reticulum, Golgiapparatus, and as a vesicle that transiently fuses at the cell plasmamembrane, releasing the proteins outside of the cell.

By “signal sequence” or “leader sequence” is meant a peptide sequence(5, 10, 15, 20, 25, 30 amino acids long) present at theN-terminus ofnewly synthesized proteins that directs their entry to the secretorypathway. Exemplary leader sequences include the kappa leader sequence:METDTLLLWVLLLWVPGSTGD [SEQ ID NO:15] (human), METDTLLLWVLLLWVPGSTGD [SEQID NO:16] (mouse); and the CDS leader sequence: MALPVTALLLPLALLLHAARP[SEQ ID NO:17].

By “soluble” is meant a polypeptide that is freely diffusible in anaqueous environment (e.g., not membrane bound).

By “specifically binds” is meant a polypeptide or fragment thereof thatrecognizes and binds a biological molecule of interest (e.g., apolypeptide), but which does not substantially recognize and bind othermolecules in a sample, for example, a biological sample, which naturallyincludes a polypeptide of the invention.

The term “tumor antigen” as used herein refers to an antigen (e.g., apolypeptide) that is uniquely or differentially expressed on a tumorcell compared to a normal or non-IS neoplastic cell. With reference tothe invention, a tumor antigen includes any polypeptide expressed by atumor that is capable of activating or inducing an immune response viaan antigen recognizing receptor (e.g., CD19, MUCI) or capable ofsuppressing an immune response via receptor-ligand binding (e.g., CD47,PD-L1/L2, B7.1/2).

By “virus antigen” is meant a polypeptide expressed by a virus that iscapable of inducing an immune response.

The terms “comprises”, “comprising”, and are intended to have the broadmeaning ascribed to them in U.S. Patent Law and can mean “includes”,“including” and the like.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the disease course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Therapeutic effects of treatment include,without limitation, preventing occurrence or recurrence of disease,alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease, preventing metastases,decreasing the rate of disease progression, amelioration or palliationof the disease state, and remission or improved prognosis. By preventingprogression of a disease or disorder, a treatment can preventdeterioration due to a disorder in an affected or diagnosed subject or asubject suspected of having the disorder, but also a treatment mayprevent the onset of the disorder or a symptom of the disorder in asubject at risk for the disorder or suspected of having the disorder.

The term “subject” as used herein refers to a vertebrate, preferably amammal, more preferably a human.

The term “immunocompromised” as used herein refers to a subject who hasan immunodeficiency. The subject is very vulnerable to opportunisticinfections, infections caused by organisms that usually do not causedisease in a person with a healthy immune system, but can affect peoplewith a poorly functioning or suppressed immune system.

Other aspects of the invention are described in the following disclosureand are within the ambit of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying drawings.

FIG. 1 depicts T cells modified to express the chimeric antigen receptor(CAR) alone or in combination with secretable scFv (e.g. αPD-1, αPD-L1,αCTLA-4, or αCD47). T cells modified to express the chimeric antigenreceptor (CAR) alone are subject to suppression within the hostile tumormicroenvironment. Without being bound to a particular theory, furthermodification of these cells to express secretable scFv to blockimmunosuppressive signaling has improved anti-tumor function due totheir ability to modulate the tumor microenvironment and resistsuppressive factors.

FIGS. 2A and 2B depict the structure of secretable anti-CD47 scFvconstructs. FIG. 2A depicts the structure of a secretable anti-CD47 scFvdesigned to include a kappa (κ) leader sequence to allow exportation ofthis protein. The variable heavy (V_(H)) and light (V_(L)) chains werelinked with a serine glycine linker (G₄S) (SEQ ID NO: 52) and a myc-tagpeptide was included to allow detection of the scFv. FIG. 2B depicts thesecretable scFv was linked to the 1928z CAR construct using a P2Aelement as shown.

FIG. 3 depicts the B6H12.2 scFv sequence operably linked to a Kappaleader sequence. The variable heavy (V_(H)) and variable light (V_(L))sequences of the B6H12.2 hybridoma were PCR amplified with a kappaleader sequence, a c-myc tag and joined with a serine glycine linker.The nucleic acid sequence (SEQ ID NO: 18) and amino acid translation(SEQ ID NO: 53) are shown.

FIG. 4 depicts B6H12.2 scFv sequence operably linked to a CDS leadersequence. The variable heavy (V_(H)) and variable light (V_(L))sequences of the B6H12.2 hybridoma were PCR amplified with a CDS leadersequence, a c-myc tag and joined with a serine glycine linker. Thenucleic acid sequence (SEQ ID NO: 19) and amino acid translation (SEQ IDNO: 54) are shown.

FIG. 5 depicts the nucleic acid sequence of the 1928z-2A-B6H12.2 (kappaleader) construct [SEQ ID NO:20]. The B6H12.2 scFv sequence was clonedinto an SFG expression vector for expression with the CD19-targeted1928z CAR. A P2A element was used to join the two elements, as shown.

FIG. 6 depicts the nucleic acid sequence of the 4H1128z-2A-B6H12.2(kappa leader) construct [SEQ ID NO:21]. The B6H12.2 scFv sequence wascloned into an SFG expression vector for expression with theMUC-CD-targeted 4H1128z chimeric antigen receptor (CAR). A P2A elementwas used to join the two elements, as shown.

FIGS. 7A and 7B depict the generation of 1928-2A-B6H12.2 293G1v9packaging cells. Viral packaging cells were generated using the1928z-2A-B6H12.2 or 1928z vector. FIG. 7A depicts selection of twoclones, clones 5 and 6, based on expression of 1928z CAR, which wascomparable to control 1928z 293G1v9 cells. CAR expression was determinedby flow cytometry and staining with 12dll antibody. FIG. 7B depicts anexperiment where supernatant from 1928z or 1928z-2A-B6H12.2 packagingcells was incubated with CD47⁺ tumor cells, Nalm-6 and Raji, and thetumor cells were washed and stained with anti-CD47. Tumor cellsincubated in 1928z-2A-B6H12.2 supernatant had decreased anti-CD47binding compared to incubation with 1928z supernatant. Supernatant fromthe B6H12.2 hybridoma cells was used as a control.

FIGS. 8A and 8B depict the generation of 1928z-2A-B6H12.2 humanperipheral blood T cells. Human peripheral blood T cells were transducedwith supernatant from 1928z or 1928z-2A-B6H12.2 packaging cells. FIG. 8Adepicts analysis by flow cytometry of CAR expression using the 12d11antibody and of bound anti-CD47 scFv stained with a fluorescently taggedanti-c-myc tag antibody. FIG. 8B depicts the ability of the anti-CD47scFv to block CD47, determined by staining T cells with anti-CD47antibody. 1928z-2A-B6H12.2 T cells had decreased anti-CD47 bindingcompared to 1928z T cells. 1928z T cells incubated in B6H12.2 hybridomasupernatant were used as a control.

FIGS. 9A-9C depict 1928z-2A human peripheral blood T cells. Flowcytometry was performed to characterize the phenotype of 1928z and1928z-2A-B6H12.2 T cells. FIG. 9A depicts that 1928z and 1928z-2A Tcells had an equivalent ratio of CD4:CD8 T cells, and equivalentexpression of activation markers CD69 and CD25. 1928z T cells hadincreased expression of CD62L compared to 1928z-2A-B6H12.2 T cells. FIG.9B depicts the ability of 1928z and 1928z-2A-B6H12.2 T cells to secretecytokines, as assessed by flow cytometry following incubation with 3T3(CD19⁺/B7.1⁺) aAPCs cells and golgi transport inhibitors, Golgi plug andGolgi Stop. 1928z and 1928z-2A-B6H12.2 T cells produced equivalentlevels of IL-2 and IFNg following stimulation with 3T3(CD19⁺/B7.1⁺)cells. FIG. 9C depicts that 1928z and 1928z-2A-B6H12.2 T cells haveequivalent cytolytic capacity, as determined by a standard ⁵¹Chromiumrelease assay using Raji tumor cells.

FIGS. 10A and 10B depict the anti-tumor efficacy of 1928z-2A-B6H12.2 Tcells. The in vivo anti-tumor efficacy of 1928z-2A-B6H12.2 T cells wasinvestigated with a preclinical SCID-Beige mouse model. Mice wereintravenously inoculated with 1×10⁶ Nalm-6-FireFly luciferase⁺ tumorcells and subsequently treated with 5.7×10⁶ CAR⁺ 1928 z,1928z-2A-B6H12.2 or control ovarian cancer targeted 4H1128z-2A-B6H12.2 Tcells, also inoculated intravenously. FIG. 10A depicts that mice treatedwith 1928z-2A-B6H12.2 T cells had enhanced survival compared tountreated, 1928z or 4H1128z-2A-B6H12.2 treated mice. FIG. 10B depictsthat 1928z-2A-B6H12.2 treated mice have reduced tumor burden compared tonontreated, 1928z or 4H1128z-2A-B6H12.2 T cell treated mice, usingbioluminescent imaging to monitor tumor progression.

FIG. 11 depicts the 5C4 scFv sequence operably linked to a Kappa leadersequence. The variable heavy (V_(H)) and variable light (V_(L))sequences of the 5C4 antibody clone were designed with the kappa leadersequence, a c-myc tag and joined with a serine glycine linker. Thenucleic acid sequence (SEQ ID NO: 22) and amino acid translation (SEQ IDNO: 55) are shown.

FIG. 12 depicts the nucleic acid sequence of the 1928z-2A-5C4 (kappaleader) construct [SEQ ID NO:23]. The 5C4 scFv sequence was cloned intoan SFG expression vector for expression with the CD19-targeted 1928zCAR. A P2A element was used to join the two elements, as shown.

FIG. 13 depicts the nucleic acid sequence of the 4H1128z-2A-5C4 (kappaleader) construct [SEQ ID NO:24]. The 5C4 scFv was cloned into an SFGexpression vector to be expressed with the MUC-CD-targeted 4H1128z CAR.A P2A element was used to join the two elements, as shown.

FIG. 14 depicts the generation of 1928z-2A-5C4 (Kappa Leader) 293G1v9cells. Viral packaging cells were generated using the 1928z-2A-5C4. Twoclones, clones A6 and B6, were selected based on expression of the 1928zCAR, which was comparable to control1928z 293G1v9 cells. CAR expressionwas determined by flow cytometry and staining with 12d11 antibody.

FIG. 15 depicts the generation of 1928z-2A-5C4 (kappa leader) humanperipheral blood T cells. Human peripheral blood T cells were transducedwith supernatant from 1928z or 1928z-2A-5C4 packaging cells. Flowcytometry was used to analyze CAR expression using the 12d11 antibodyand of bound anti-CD47 scFv using staining with a fluorescently taggedanti-c-myc tag antibody.

FIGS. 16A-16C depict PD-L1 expression on 3T3(CD19⁺/B7.1⁺), Raji andNalm-6 cells. Flow cytometry was used to determine expression of PD-L1on 3T3 (CD19⁺/B7.1⁺), Raji and Nalm-6 cells that had been transduced toexpress PD-L1. Transduced cells expressed significant levels of PD-L1compared to control untransduced cells and circled populations weresorted for use in experiments.

FIG. 17 depicts 1928z and 1928z-2A-5C4 T cell expansion. 1928z and1928z-2A-5C4 T cells were incubated with 3T3(CD19⁺/B7.1⁺) or3T3(CD19⁺/B7.1⁺/PD-L1⁺), T cell expansion was monitored with Trypan blueand CAR expression was determined by flow cytometry. Expansion and CARexpression was correlated to that of cells expanded on 3T3 (CD19⁺/B7.1⁺)cells.

FIG. 18 depicts the J43 scFv sequence operably linked to a mouse kappaleader sequence. The variable heavy (VH) and variable light (VL)sequences of the J43 antibody clone was designed with the mouse kappaleader sequence, a c-myc tag and joined with a serine glycine linker.The nucleotide sequence (SEQ ID NO: 25) and amino acid translation (SEQID NO: 56) are shown.

FIG. 19 depicts the nucleic acid sequence of the 19m28mziRESJ43 (mousekappa leader) construct [SEQ ID NO:26]. The J43 scFv was cloned into anSFG expression vector for expression with the CD19-targeted 19m28mz CAR.An internal ribosome entry site (IRES) element was used to join the twoelements, as shown.

FIG. 20 depicts the nucleic acid sequence of the 4H11m28mziRESJ43 (mousekappa leader) construct [SEQ ID NO:27]. The J43 scFv was cloned into anSFG expression vector for expression with the MUC-CD-targeted 4H11m28mzCAR. An internal ribosome entry site (IRES) element was used to join thetwo elements, as shown.

FIG. 21 depicts strategies to genetically modify CART cells to expressscFv molecules (“armored CAR T cells”) to overcome “hostile” tumormicroenvironment. CAR⁺ T cells may be modified to secrete antagonisticscFvs with immune regulatory functions. Upon activation of the CAR tocognate antigen (1), armored CAR modified T cells may be induced tosecrete scFvs antagonistic to the inhibitory PD-1 T cell receptor onboth infused CAR modified T cells and endogenous anti-tumor T cellsenhancing anti-tumor effector function (2), induced to secrete scFvsantagonistic to the inhibitory CTLA-4 T cell receptor on both infusedCAR modified T cells and endogenous anti-tumor T cells enhancinganti-tumor effector function (3), or induced to secrete an scFvantagonistic to the CD47 receptor expressed on the tumor cell reversingthe cloaking the tumor cell from recognition by the host innateanti-tumor immune response leading to recognition and eradication oftumor by host macrophages.

FIG. 22A-22D depict constitutive expression of CD40L by human T-cells.(A) Schematic of retroviral construct encoding human CD40L vector; LTR,long terminal repeat; SD, SA, splice donor and acceptor; Ψ, packagingelement. (B) Flow cytometry of CD4+ and CD8+CD40L-modified T-cellsfollowing retroviral gene transfer; x-axis APC-conjugated anti-humanCD40L (CD154). (C) Enhanced proliferation of CD40L-modified T-cellscompared to mock transduced T-cells. (D) Enhanced secretion of solubleCD40L (sCD40L), IFN-γ, and GM-CSF of CD40L-modified T-cells compared tomock transduced T-cells. All results are representative of at leastthree separate experiments. (* denotes statistical significance)

FIGS. 23A and 23 B depict augmented immunogenicity of CD40+ Tumor cellsby CD40L-modified T-cells. (A) Flow cytometry showing upregulation ofco-stimulatory molecules (CD80 and CD86), adhesion molecules (CD54,CD58, and CD70) HLA molecules (HLA Class I and HLA-DR), and theFas-death receptor (CD95) on DOHH2 tumor cell line following co-culturewith CD40L-modified T-cells (solid line) compared to culture withmock-transduced T-cells from the same donor (gray line). (B) CD40-tumor(NALM6 shown) demonstrating no phenotypic changes following co-culturewith CD40L-modified T-cells. All results are representative of at leastthree separate experiments.

FIGS. 24A and 24B depict augmented immunogenicity of CLL cells byautologous CD40L-modified T-cells. (A) Flow cytometry of patient derivedCD40L-modified T-cells following retroviral gene transfer with CD40Lcontaining retroviral vector; x-axis APC-conjugated anti-human CD40L(CD154). (B) Flow cytometry showing upregulation of co-stimulatorymolecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70) HLAmolecules (HLA Class I and HLA-DR), and the Fas-death receptor (CD95) onCLL cells after co-culturing with autologous CD40L-modified T-cells(solid line) compared to co-cultures with mock-transduced T-cells fromthe same donor (gray line). All results are representative of at leastthree separate experiments.

FIGS. 25A and 25B depict secretion of IL-12 and maturation of monocytederived Dendritic Cells (moDCs) by CD40L-modified T-cells. (A) Cytokineanalysis of culture media for co-cultures (24 hours) between moDCs andCD40L-modified T-cells from three separate donors demonstrating elevatedIL-12p70 secretion. (B) Flow cytometry of moDCs demonstrating maturationfollowing co-culture with CD40L-modified T-cells. All results arerepresentative of at least three experiments.

FIG. 26A-26C depict efficient transduction of human T-cells with aCAR/CD40L vector demonstrates enhanced cytotoxicity. (A) Schematic ofretroviral construct containing 1928z-IRES-CD40L and Pz1-IRES-CD40Lgenes; LTR, long terminal repeat; SD, SA, splice donor and acceptor; Ψ,packaging element; CD8 indicates CD8 leader sequence; scFv, single chainvariable fragment; VH and VL, variable heavy and light chains; TM,transmembrane domain. (B) FACS analysis of human T-cells transduced toexpress 19-28z/CD40L vector (pre-stimulation) with subsequent enhancedexpression of CAR/CD40L following co-culture on AAPCs (NIH 3T3fibroblasts expressing CD19 and CD80; 1928/CD40LT-cells shown) used forin vivo experiments. x-axis, PE-conjugated 1928z CAR-specific antibody(19e3); y-axis, APC-conjugated anti-human CD40L (CD154). (C) Asdetermined by standard 51Cr release assay 19-28z/40L T-cells havesignificant increased ability to lyse DOHH2 tumor cells compared to19-28z T-cells. All results are representative of at least threeexperiments. (* denotes statistical significance).

FIG. 27 depicts tumor eradication and long term survival following1928z/CD40L T-cell infusion. Survival curve of SCID-Beige miceinoculated with DOHH2 tumor cells by intravenous (i.v.) injection 2 daysbefore a single i.v. dose of CAR-modified T-cells. Enhanced long-termsurvival was demonstrated in mice treated with 1928z/CD40L T-cells(n=10) as compared to a panel of control T cells (1928z group n=8; Pz1and Pz1/40L group n=5). Results are representative of at least twoexperiments. (* denotes statistical significance).

FIG. 28 depicts augmented immunogenicity of CD40+ Tumor cells by sCD40L.(A) Flow cytometry showing upregulation of co-stimulatory molecule(CD80), adhesion molecules (CD54, CD58, and CD70) HLA molecules (HLAClass I and HLA-DR), and the Fas-death receptor on DOHH2 tumor cell linefollowing co-culture with conditioned media (CD40L-modified T-cellsmedia) containing elevated levels of sCD40L (solid line) compared tomedia (mock-transduced T-cells media) without elevated levels of sCD40L(gray line).

FIG. 29 depicts 1928z/CD40L T-cells demonstrates enhanced cytotoxicity.As determined by standard 51Cr release assay 19-28z/40L T-cells havesignificant increased ability to lyse Raji tumor cells compared to19-28z T-cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally provides cells, including geneticallymodified immunoresponsive cells (e.g., T cells, Natural Killer (NK)cells, cytotoxic T lymphocytes (CTL) cells) expressing at least acombination of an antigen-recognizing receptor (e.g., TCR or CAR) andeither (i) an scFv that binds an immunosuppressive antigen (e.g. aPD-1,αPD-L1, αCTLA-4, or αCD47)); (ii) an scFv that binds animmunostimulatory antigen (e.g. αCD28, αOX-40, αCD40 or α4-1BB) or (iii)CD40L, and methods of using such cells for the treatment of neoplasiaand other pathologies where an increase in an antigen-specific immuneresponse is desired. The invention is based, at least in part, on thediscovery that scFvs that bind an immunosuppressive antigen (e.g. CD47and PD-L1 as shown herein) are useful for activating and stimulating animmunoreactive cell. In particular, the scFvs of the invention decreaseor prevent suppression of the immune response of an activatedimmunoreactive cell in the tumor microenvironment. Malignant cells havedeveloped a series of mechanisms to protect themselves from immunerecognition and elimination. The present approach providesimmunogenicity within the tumor microenvironment for tumor eradication,and represents a significant advance over conventional adoptive T celltherapy.

Tumor Microenvironment

Tumors have a microenvironment that is hostile to the host immuneresponse involving a series of mechanisms by malignant cells to protectthemselves from immune recognition and elimination. This “hostile tumormicroenvironment” comprises a variety of immune suppressive factorsincluding infiltrating regulatory CD4⁺ T cells (Tregs), myeloid derivedsuppressor cells (MDSCs), tumor associated macrophages (TAMs), immunesuppressive cytokines including IL-10 and TGF-β, and expression ofligands targeted to immune suppressive receptors expressed by activatedT cells (CTLA-4 and PD-1). These mechanisms of immune suppression play arole in the maintenance of tolerance and suppressing inappropriateimmune responses, however within the tumor microenvironment thesemechanisms prevent an effective anti-tumor immune response. Collectivelythese immune suppressive factors can induce either marked anergy orapoptosis of adoptively transferred CAR modified T cells upon encounterwith targeted tumor cells.

Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4)

CTLA-4 is an inhibitory receptor expressed by activated T cells, whichwhen engaged by its corresponding ligands (CD80 and CD86; B7-1 and B7-2,respectively), mediates activated T cell inhibition or anergy. In bothpreclinical and clinical studies, CTLA-4 blockade by systemic antibodyinfusion, enhanced the endogenous anti-tumor response albeit, in theclinical setting, with significant unforeseen toxicities. Without beingbound to a particular theory, targeted CTLA-4 blockade through deliveryof antagonistic scFvs by tumor targeted CAR modified T cells allows forreduced toxicity as well as provides a surrogate “endogenous” populationof tumor targeted T cells (the CART cell population) protected fromimmune suppression. Pre-clinical studies (e.g., human xenograft tumormodels and murine tumor models of B cell malignancies and ovariancarcinomas) can be used to evaluate the effect of scFv secretion both onthe CAR modified T cell population as well as on the endogenousanti-tumor immune response. Anti-CTLA-4 scFv can be generated from the9D9 hybridoma, which secretes mouse anti-mouse CTLA-4 monoclonalantibodies, or the 9H10 hybridoma, which secretes hamster anti-mouseCTLA-4 monoclonal antibodies.

Programmed Cell Death Protein 1 (PD-1)

PD-1 is a negative immune regulator of activated T cells upon engagementwith its corresponding ligands PD-L1 and PD-L2 expressed on endogenousmacrophages and dendritic cells. Upregulation of PD-L1 is one mechanismtumor cells may evade the host immune system. Again, in bothpre-clinical and recently published clinical trials, PD-1 blockade byantagonistic antibodies induced anti-tumor responses mediated throughthe host endogenous immune system. Xenograft and syngeneic murine tumormodels can be used to show that antagonistic anti-PD-1 scFvs secreted bytumor targeted CAR modified T cells enhance the anti-tumor efficacy ofthese scFv secreting CAR modified T cells.

CD47

CD47 is a membrane protein with broad tissue distribution and one whichhas been shown in recent preclinical imodels to protect a wide array oftumor cells from macrophage recognition. In these models, infusion ofanti-CD47 monoclonal antibodies resulted in a decrease of establishedtumor progression. In other words, CD47 blockade on tumor cells exposedthese tumor cells to recognition and phagocytosis by the hostmacrophages. Given the rather ubiquitous expression of this antigen,systemic blocking antibody infusion may potentially lead to off-targettoxicity. Again, in keeping with the paradigm of targeted delivery,secretion of similarly blocking anti-CD47 scFvs delivered directly tothe tumor microenvironment by CAR modified T cells induce/enhance adesired anti-tumor effect, in this case mediated by the innate ratherthan adaptive host immune system. Furthermore, this approach is notlimited to the treatment of neoplasias, but is amenable to a wide rangeof applications where an increase in an antigen-specific immune responseis desired, such applications include not only the treatment ofneoplasias, but also for the enhancement of an immune response against apathogen infection or an infectious disease and to reinforce immunetolerance in regulatory T cells in the context of autoimmunity orallogeneic transplantation.

CD40L

CD40 ligand (CD40L, CD154), a type II transmembrane protein belonging tothe tumor necrosis factor (TNF) gene superfamily, has the potential toenhance tumor specific T-cell function. Initially identified onactivated CD4+ T-cells, expression of CD40L is inducible on a vast arrayof immune, hematopoietic, epithelial, endothelial and smooth musclecells. In activated T-cells, CD40L is expressed within minutes, peakingwithin 6 hours, and then declining over the subsequent 12-24 hours.CD40L binds to its cognate receptor CD40 which is constitutivelyexpressed on a variety of immune and non-immune cells including B-cells,macrophages, and dendritic cells (DCs). Significantly, CD40 is alsoexpressed on several hematologic and non-hematologic malignanciesincluding chronic lymphocytic leukemia (CLL), acute lymphoblasticleukemia (ALL), non-Hodgkin lymphoma (NHL), Hodgkin Lymphoma,nasopharyngeal carcinoma, osteosarcoma, Ewing sarcoma, melanoma, breast,ovarian, and cervical carcinoma demonstrating potential application ofCAR/CD40L T-cells to a broad array of malignancies. See references 8-17listed in the references to Example 6, below.

Hematopoietic Cell Lineages

Mammalian hematopoietic (blood) cells provide a diverse range ofphysiologic activities. Hematopoietic cells are divided into lymphoid,myeloid and erythroid lineages. The lymphoid lineage, comprising B, Tand natural killer (NK) cells, provides for the production ofantibodies, regulation of the cellular immune system, detection offoreign agents in the blood, detection of cells foreign to the host, andthe like. The term “T cells” as used herein refers to lymphocytes thatmature in the thymus and are chiefly responsible for cell-mediatedimmunity. T cells are involved in the adaptive immune system. The term“natural killer (NK) cells” as used herein refers to lymphocytes thatare part of cell-mediated immunity and act during the innate immuneresponse. They do not require prior activation in order to perform theircytotoxic effect on target cells. Cytotoxic T cells (CTL or killer Tcells) are a subset of T lymphocytes capable of inducing the death ofinfected somatic or tumor cells.

Cells for Use in the Methods of the Invention

The present invention provides cells expressing a combination of anantigen-recognizing receptor that activates an immunoresponsive cell(e.g., TCR, CAR) and an scFv that binds an immunosuppressive antigen(e.g. αPD-1, αPD-L1, αCTLA-4, or αCD47), and methods of using such cellsfor the treatment of a disease that requires an enhanced immuneresponse. In one approach, tumor antigen-specific T cells, NK cells, CTLcells or other immunoresponsive cells are used to express an scFv thatbinds an immunosuppressive antigen, for the treatment or prevention ofneoplasia. For example, a T cell expressing a chimeric antigen receptor1928z that recognizes CD19 is co-expressed in a T cell that expresses anscFv that binds CD47. Such cells are administered to a human subject inneed thereof for the treatment or prevention of blood cancers (e.g.leukemias, lymphomas, and myelomas). In another approach, viralantigen-specific T cells, NK cells, CTL cells can be used for thetreatment of viral diseases. For example, a chimeric co-stimulatoryantigen receptor that recognizes a first CMV antigen and an scFv thatbinds PD-1 are co-expressed in cytotoxic T lymphocytes for the treatmentof CMV.

A patient's own T cells may be genetically modified to target tumorsthrough the introduction of genes encoding artificial T cell receptorstermed chimeric antigen receptors (CARs). First generation CARs aretypically composed of an antibody-derived antigen recognition domain, asingle fragment length antibody (scFv), fused to a variabletrans-membrane domain, fused to cytoplasmic signaling domain of the Tcell receptor chain. Additional inclusion of one or two co-stimulatoryreceptor signaling domains including CD28, 4-1BB, and OX-40 proximal tothe C chain enhances CAR signaling resulting in second and thirdgeneration CARs respectively.

Tumor Antigen-Specific T Lymphocytes (and NK Cells)

Types of tumor antigen-specific human lymphocytes that can be used inthe methods of the invention include, without limitation, peripheraldonor lymphocytes genetically modified to express chimeric antigenreceptors (CARs) (Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45),peripheral donor lymphocytes genetically modified to express afull-length tumor antigen-recognizing T cell receptor complex comprisingthe a and β heterodimer (Morgan, R. A., et al. 2006 Science314:126-129), lymphocyte cultures derived from tumor infiltratinglymphocytes (TILs) in tumor biopsies (Panelli, M. C., et al. 2000 JImmunol 164:495-504; Panelli, M. C., et al. 2000 J Immunol164:4382-4392), and selectively in vitro-expanded antigen-specificperipheral blood leukocytes employing artificial antigen-presentingcells (AAPCs) or pulsed dendritic cells (Dupont, J., et al. 2005 CancerRes 65:5417-5427; Papanicolaou, G. A., et al. 2003 Blood 102:2498-2505).The T cells may be autologous, allogeneic, or derived in vitro fromengineered progenitor or stem cells. Any suitable tumor antigen(antigenic peptide) is suitable for use in the tumor-related embodimentsdescribed herein. Sources of antigen include, but are not limited tocancer proteins. The antigen can be expressed as a peptide or as anintact protein or portion thereof. The intact protein or a portionthereof can be native or mutagenized.

Suitable antigens include prostate specific membrane antigen (PSMA) andprostate stem cell antigen (PCSA).

Viral Antigen-Specific T Lymphocytes (and NK Cells)

Suitable antigens for use in the treatment of pathogen infection orother infectious disease, for example, in an immunocompromised subjectinclude, without limitation, viral antigens present in Cytomegalovirus(CMV), Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), andinfluenza virus.

The unpurified source of CTLs may be any known in the art, such as thebone marrow, fetal, neonate or adult or other hematopoietic cell source,e.g., fetal liver, peripheral blood or umbilical cord blood. Varioustechniques can be employed to separate the cells. For instance, negativeselection methods can remove non-CTLs initially. mAbs are particularlyuseful for identifying markers associated with particular cell lineagesand/or stages of differentiation for both positive and negativeselections.

A large proportion of terminally differentiated cells can be initiallyremoved by a relatively crude separation. For example, magnetic beadseparations can be used initially to remove large numbers of irrelevantcells. Preferably, at least about 80%, usually at least 70% of the totalhematopoietic cells will be removed prior to cell isolation.

Procedures for separation include, but are not limited to, densitygradient centrifugation; resetting; coupling to particles that modifycell density; magnetic separation with antibody-coated magnetic beads;affinity chromatography; cytotoxic agents joined to or used inconjunction with a mAb, including, but not limited to, complement andcytotoxins; and panning with antibody attached to a solid matrix, e.g.plate, chip, elutriation or any other convenient technique.

Techniques for separation and analysis include, but are not limited to,flow cytometry, which can have varying degrees of sophistication, e.g.,a plurality of color channels, low angle and obtuse light scatteringdetecting channels, impedance channels.

The cells can be selected against dead cells, by employing dyesassociated with dead cells such as propidium iodide (PI). Preferably,the cells are collected in a medium comprising 2% fetal calf serum (FCS)or 0.2% bovine serum albumin (BSA) or any other suitable, preferablysterile, isotonic medium.

Accordingly, the invention generally provides an immunoresponsive cell,such as a virus specific or tumor specific T cell comprising a receptorthat binds a first antigen and activates the immunoresponsive cell and areceptor that binds a second antigen and stimulates the immunoresponsivecell.

Vectors

Genetic modification of immunoresponsive cells (e.g., T cells, CTLcells, NK cells) can be accomplished by transducing a substantiallyhomogeneous cell composition with a recombinant DNA construct.Preferably, a retroviral vector (either gamma-retroviral or lentiviral)is employed for the introduction of the DNA construct into the cell. Forexample, a polynucleotide encoding a receptor that binds an antigen(e.g., a tumor antigen, or a variant, or a fragment thereof), can becloned into a retroviral vector and expression can be driven from itsendogenous promoter, from the retrovirallong terminal repeat, or from apromoter specific for a target cell type of interest. Non-viral vectorsmay be used as well.

For initial genetic modification of the cells to provide tumor or viralantigen-specific cells, a retroviral vector is generally employed fortransduction, however any other suitable viral vector or non-viraldelivery system can be used. For subsequent genetic modification of thecells to provide cells comprising an antigen presenting complexcomprising at least two co-stimulatory ligands, retroviral gene transfer(transduction) likewise proves effective. Combinations of retroviralvector and an appropriate packaging line are also suitable, where thecapsid proteins will be functional for infecting human cells. Variousamphotropic virus-producing cell lines are known, including, but notlimited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437);PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP(Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464).Non-amphotropic particles are suitable too, e.g., particles pseudotypedwith VSVG, RD114 or GALV envelope and any other known in the art.

Possible methods of transduction also include direct co-culture of thecells with producer cells, e.g., by the method of Bregni, et al. (1992)Blood 80:1418-1422, or culturing with viral supernatant alone orconcentrated vector stocks with or without appropriate growth factorsand polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat.22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

Other transducing viral vectors can be used to express a co-stimulatoryligand of the invention in an immunoresponsive cell. Preferably, thechosen vector exhibits high efficiency of infection and stableintegration and expression (see, e.g., Cayouette et al., Human GeneTherapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844,1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini etal., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad.Sci. U.S.A. 94:10319, 1997). Other viral vectors that can be usedinclude, for example, adenoviral, lentiviral, and adena-associated viralvectors, vaccinia virus, a bovine papilloma virus, or a herpes virus,such as Epstein-Barr Virus (also see, for example, the vectors ofMiller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281,1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al.,Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research andMolecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984;Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; andJohnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularlywell developed and have been used in clinical settings (Rosenberg etal., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No.5,399,346).

Non-viral approaches can also be employed for the expression of aprotein in cell. For example, a nucleic acid molecule can be introducedinto a cell by administering the nucleic acid in the presence oflipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413,1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am.J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al.,Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal ofBiological Chemistry 264:16985, 1989), or by micro-injection undersurgical conditions (Wolff et al., Science 247:1465, 1990). Othernon-viral means for gene transfer include transfection in vitro usingcalcium phosphate, DEAE dextran, electroporation, and protoplast fusion.Liposomes can also be potentially beneficial for delivery of DNA into acell. Transplantation of normal genes into the affected tissues of asubject can also be accomplished by transferring a normal nucleic acidinto a cultivatable cell type ex vivo (e.g., an autologous orheterologous primary cell or progeny thereof), after which the cell (orits descendants) are injected into a targeted tissue or are injectedsystemically. Recombinant receptors can also be derived or obtainedusing transposases or targeted nucleases (e.g. Zinc finger nucleases,meganucleases, or TALE nucleases). Transient expression may be obtainedby RNA electroporation.

cDNA expression for use in polynucleotide therapy methods can bedirected from any suitable promoter (e.g., the human cytomegalovirus(CMV), simian virus 40 (SV40), or metallothionein promoters), andregulated by any appropriate mammalian regulatory element or intron(e.g. the elongation factor 1a enhancer/promoter/intron structure). Forexample, if desired, enhancers known to preferentially direct geneexpression in specific cell types can be used to direct the expressionof a nucleic acid. The enhancers used can include, without limitation,those that are characterized as tissue- or cell-specific enhancers.Alternatively, if a genomic clone is used as a therapeutic construct,regulation can be mediated by the cognate regulatory sequences or, ifdesired, by regulatory sequences derived from a heterologous source,including any of the promoters or regulatory elements described above.

The resulting cells can be grown under conditions similar to those forunmodified cells, whereby the modified cells can be expanded and usedfor a variety of purposes.

Polypeptides and Analogs

Also included in the invention are aCD19, CD28, CD3ζ, 4H1128z, B6H12.2scFv, 5C4 scFv, and J43 scFv polypeptides or fragments thereof that aremodified in ways that enhance their anti-neoplastic activity (e.g., ahumanized monoclonal antibody) when expressed in an immunoresponsivecell. The invention provides methods for optimizing an amino acidsequence or nucleic acid sequence by producing an alteration in thesequence. Such alterations may include certain mutations, deletions,insertions, or post-translational modifications. The invention furtherincludes analogs of any naturally-occurring polypeptide of theinvention. Analogs can differ from a naturally-occurring polypeptide ofthe invention by amino acid sequence differences, by post-translationalmodifications, or by both. Analogs of the invention will generallyexhibit at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identity with all or part of a naturally-occurring amino, acidsequence of the invention. The length of sequence comparison is at least5, 10, 15 or 20 amino acid residues, preferably at least 25, 50, or 75amino acid residues, and more preferably more than 100 amino acidresidues. Again, in an exemplary approach to determining the degree ofidentity, a BLAST program may be used, with a probability score betweene⁻³ and e⁻¹⁰⁰ indicating a closely related sequence. Modificationsinclude in vivo and in vitro chemical derivatization of polypeptides,e.g., acetylation, carboxylation, phosphorylation, or glycosylation;such modifications may occur during polypeptide synthesis or processingor following treatment with isolated modifying enzymes. Analogs can alsodiffer from the naturally-occurring polypeptides of the invention byalterations in primary sequence. These include genetic variants, bothnatural and induced (for example, resulting from random mutagenesis byirradiation or exposure to ethanemethylsulfate or by site-specificmutagenesis as described in Sambrook, Fritsch and Maniatis, MolecularCloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel etal., supra). Also included are cyclized peptides, molecules, and analogswhich contain residues other than L-amina acids, e.g., D-amino acids ornon-naturally occurring or synthetic amino acids, e.g., .beta. or.gamma. amino acids.

In addition to full-length polypeptides, the invention also providesfragments of any one of the polypeptides or peptide domains of theinvention. As used herein, the term “a fragment” means at least 5, 10,13, or 15 amino acids. In other embodiments a fragment is at least 20contiguous amino acids, at least 30 contiguous amino acids, or at least50 contiguous amino acids, and in other embodiments at least 60 to 80,100, 200, 300 or more contiguous amino acids. Fragments of the inventioncan be generated by methods known to those skilled in the art or mayresult from normal protein processing (e.g., removal of amino acids fromthe nascent polypeptide that are not required for biological activity orremoval of amino acids by alternative mRNA splicing or alternativeprotein processing events).

Non-protein analogs have a chemical structure designed to mimic thefunctional activity of a protein of the invention. Such analogs areadministered according to methods of the invention. Such analogs mayexceed the physiological activity of the original polypeptide. Methodsof analog design are well known in the art, and synthesis of analogs canbe carried out according to such methods by modifying the chemicalstructures such that the resultant analogs increase the anti-neoplasticactivity of the original polypeptide when expressed in animmunoresponsive cell. These chemical modifications include, but are notlimited to, substituting alternative R groups and varying the degree ofsaturation at specific carbon atoms of a reference polypeptide.Preferably, the protein analogs are relatively resistant to in vivodegradation, resulting in a more prolonged therapeutic effect uponadministration. Assays for measuring functional activity include, butare not limited to, those described in the Examples below.

Co-Stimulatory Ligands

The interaction with at least one co-stimulatory ligand provides anon-antigen-specific signal important for full activation of an immunecell (e.g., T cell). Co-stimulatory ligands include, without limitation,tumor necrosis factor (TNF) ligands, cytokines (such as IL-2, IL-12,IL-15 or IL21), and immunoglobulin (Ig) superfamily ligands. Tumornecrosis factor (TNF) is a cytokine involved in systemic inflammationand stimulates the acute phase reaction. Its primary role is in theregulation of immune cells. Tumor necrosis factor (TNF) ligands share anumber of common features. The majority of the ligands are synthesizedas type II transmembrane proteins (extracellular C-terminus) containinga short cytoplasmic segment and a relatively long extracellular region.TNF ligands include, without limitation, nerve growth factor (NGF),CD40L (CD40L)/CD154, CD137L/4-1BBL, tumor necrosis factor alpha (TNFα),CD134L/OX40L/CD252, CD27L/CD70, Fas ligand (FasL), CD30L/CD153, tumornecrosis factor beta (TNFβ)/lymphotoxin-alpha (LTα), lymphotoxin-beta(LTβ), CD257/B cell-activating factor (BAFF)/Blys/THANK/Ta11-1,glucocorticoid-induced TNF Receptor ligand (GITRL), and TNF-relatedapoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin(Ig) superfamily is a large group of cell surface and soluble proteinsthat are involved in the recognition, binding, or adhesion processes ofcells. These proteins share structural features withimmunoglobulins—they possess an immunoglobulin domain (fold).Immunoglobulin superfamily ligands include, without limitation, CD80 andCD86, both ligands for CD28.

Administration

Compositions comprising genetically modified immunoresponsive cells ofthe invention (e.g., T cells, NK cells, CTL cells, or their progenitors)can be provided systemically or directly to a subject for the treatmentof a neoplasia, pathogen infection, or infectious disease. In oneembodiment, cells of the invention are directly injected into an organof interest (e.g., an organ affected by a neoplasia). Alternatively,compositions comprising genetically modified immunoresponsive cells areprovided indirectly to the organ of interest, for example, byadministration into the circulatory system (e.g., the tumorvasculature). Expansion and differentiation agents can be provided priorto, during or after administration of the cells to increase productionof T cells, NK cells, or CTL cells in vitro or in vivo.

The modified cells can be administered in any physiologically acceptablevehicle, normally intravascularly, although they may also be introducedinto bone or other convenient site where the cells may find anappropriate site for regeneration and differentiation (e.g., thymus).Usually, at least 1×10⁵ cells will be administered, eventually reaching1×10¹⁰ or more. Genetically modified immunoresponsive cells of theinvention can comprise a purified population of cells. Those skilled inthe art can readily determine the percentage of genetically modifiedimmunoresponsive cells in a population using various well-known methods,such as fluorescence activated cell sorting (FACS). Preferable ranges ofpurity in populations comprising genetically modified immunoresponsivecells are about 50 to about 55%, about 55 to about 60%, and about 65 toabout 70%. More preferably the purity is about 70 to about 75%, about 75to about 80%, about 80 to about 85%; and still more preferably thepurity is about 85 to about 90%, about 90 to about 95%, and about 95 toabout 100%. Dosages can be readily adjusted by those skilled in the art(e.g., a decrease in purity may require an increase in dosage). Thecells can be introduced by injection, catheter, or the like. If desired,factors can also be included, including, but not limited to,interleukins, e.g. IL-2, IL-3, IL-6, IL-11, IL7, IL12, IL1S, IL21, aswell as the other interleukins, the colony stimulating factors, such asG-, M- and GM-CSF, interferons, e.g., gamma.-interferon anderythropoietin.

Compositions of the invention include pharmaceutical compositionscomprising genetically modified immunoresponsive cells or theirprogenitors and a pharmaceutically acceptable carrier. Administrationcan be autologous or heterologous. For example, immunoresponsive cells,or progenitors can be obtained from one subject, and administered to thesame subject or a different, compatible subject. Peripheral bloodderived immunoresponsive cells of the invention or their progeny (e.g.,in vivo, ex vivo or in vitro derived) can be administered via localizedinjection, including catheter administration, systemic injection,localized injection, intravenous injection, or parenteraladministration. When administering a therapeutic composition of thepresent invention (e.g., a pharmaceutical composition containing agenetically modified immunoresponsive cell), it will generally beformulated in a unit dosage injectable form (solution, suspension,emulsion).

Formulations

Compositions of the invention comprising genetically modifiedimmunoresponsive cells can be conveniently provided as sterile liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,dispersions, or viscous compositions, which may be buffered to aselected pH. Liquid preparations are normally easier to prepare thangels, other viscous compositions, and solid compositions. Additionally,liquid compositions are somewhat more convenient to administer,especially by injection. Viscous compositions, on the other hand, can beformulated within the appropriate viscosity range to provide longercontact periods with specific tissues. Liquid or viscous compositionscan comprise carriers, which can be a solvent or dispersing mediumcontaining, for example, water, saline, phosphate buffered saline,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol, and the like) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating thegenetically modified immunoresponsive cells utilized in practicing thepresent invention in the required amount of the appropriate solvent withvarious amounts of the other ingredients, as desired. Such compositionsmay be in admixture with a suitable carrier, diluent, or excipient suchas sterile water, physiological saline, glucose, dextrose, or the like.The compositions can also be lyophilized. The compositions can containauxiliary substances such as wetting, dispersing, or emulsifying agents(e.g., methylcellulose), pH buffering agents, gelling or viscosityenhancing additives, preservatives, flavoring agents, colors, and thelike, depending upon the route of administration and the preparationdesired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”,17th edition, 1985, incorporated herein by reference, may be consultedto prepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin. According to the present invention,however, any vehicle, diluent, or additive used would have to becompatible with the genetically modified immunoresponsive cells or theirprogenitors.

The compositions can be isotonic, i.e., they can have the same osmoticpressure as blood and lacrimal fluid. The desired isotonicity of thecompositions of this invention may be accomplished using sodiumchloride, or other pharmaceutically acceptable agents such as dextrose,boric acid, sodium tartrate, propylene glycol or other inorganic ororganic solutes. Sodium chloride is preferred particularly for bufferscontaining sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the agent selected. Theimportant point is to use an amount that will achieve the selectedviscosity. Obviously, the choice of suitable carriers and otheradditives will depend on the exact route of administration and thenature of the particular dosage form, e.g., liquid dosage form (e.g.,whether the composition is to be formulated into a solution, asuspension, gel or another liquid form, such as a time release form orliquid-filled form).

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert and will notaffect the viability or efficacy of the genetically modifiedimmunoresponsive cells as described in the present invention. This willpresent no problem to those skilled in chemical and pharmaceuticalprinciples, or problems can be readily avoided by reference to standardtexts or by simple experiments (not involving undue experimentation),from this disclosure and the documents cited herein.

One consideration concerning the therapeutic use of genetically modifiedimmunoresponsive cells of the invention is the quantity of cellsnecessary to achieve an optimal effect. The quantity of cells to beadministered will vary for the subject being treated. In a oneembodiment, between 10⁴ to 10¹⁰ between 10⁵ to 10⁹, or between 10⁶ and10⁸ genetically modified immunoresponsive cells of the invention areadministered to a human subject. More effective cells may beadministered in even smaller numbers. In some embodiments, at leastabout 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, and 5×10⁸ genetically modifiedimmunoresponsive cells of the invention are administered to a humansubject. The precise determination of what would be considered aneffective dose may be based on factors individual to each subject,including their size, age, sex, weight, and condition of the particularsubject. Dosages can be readily ascertained by those skilled in the artfrom this disclosure and the knowledge in the art.

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions and to beadministered in methods of the invention. Typically, any additives (inaddition to the active cell(s) and/or agent(s)) are present in an amountof 0.001 to 50% (weight) solution in phosphate buffered saline, and theactive ingredient is present in the order of micrograms to milligrams,such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1wt %, still more preferably about 0.0001 to about 0.05 wt % or about0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, andstill more preferably about 0.05 to about 5 wt %. Of course, for anycomposition to be administered to an animal or human, and for anyparticular method of administration, it is preferred to determinetherefore: toxicity, such as by determining the lethal dose (LD) andLD50 in a suitable animal model e.g., rodent such as mouse; and, thedosage of the composition(s), concentration of components therein andtiming of administering the composition(s), which elicit a suitableresponse. Such determinations do not require undue experimentation fromthe knowledge of the skilled artisan, this disclosure and the documentscited herein. And, the time for sequential administrations can beascertained without undue experimentation.

Methods of Treatment

Provided herein are methods for treating neoplasia in a subject. Alsocontemplated herein are methods for treating a pathogen infection orother infectious disease in a subject, such as an immunocompromisedhuman subject. The methods comprise administering a T cell, NK cell, orCTL cell of the invention in an amount effective to achieve the desiredeffect, be it palliation of an existing condition or prevention ofrecurrence. For treatment, the amount administered is an amounteffective in producing the desired effect. An effective amount can beprovided in one or a series of administrations. An effective amount canbe provided in a bolus or by continuous perfusion.

An “effective amount” (or, “therapeutically effective amount”) is anamount sufficient to effect a beneficial or desired clinical result upontreatment. An effective amount can be administered to a subject in oneor more doses. In terms of treatment, an effective amount is an amountthat is sufficient to palliate, ameliorate, stabilize, reverse or slowthe progression of the disease, or otherwise reduce the pathologicalconsequences of the disease. The effective amount is generallydetermined by the physician on a case-by-case basis and is within theskill of one in the art. Several factors are typically taken intoaccount when determining an appropriate dosage to achieve an effectiveamount. These factors include age, sex and weight of the subject, thecondition being treated, the severity of the condition and the form andeffective concentration of the antigen-binding fragment administered.

For adoptive immunotherapy using antigen-specific T cells, cell doses inthe range of 10⁶-10¹⁰ (e.g., 10⁹) are typically infused. Uponadministration of the genetically modified cells into the host andsubsequent differentiation, T cells are induced that are specificallydirected against the specific antigen. “Induction” of T cells caninclude inactivation of antigen-specific T cells such as by deletion oranergy. Inactivation is particularly useful to establish or reestablishtolerance such as in autoimmune disorders. The modified cells can beadministered by any method known in the art including, but not limitedto, intravenous, subcutaneous, intranodal, intratumoral, intrathecal,intrapleural, intraperitoneal and directly to the thymus.

Therapeutic Methods

The invention provides methods for increasing an immune response in asubject in need thereof. In one embodiment, the invention providesmethods for treating or preventing a neoplasia in a subject. Theinvention provides therapies that are particularly useful for thetreatment of subjects having blood cancers (e.g. leukemias, lymphomas,and myelomas) or ovarian cancer, that are not amenable to conventionaltherapeutic interventions. Suitable human subjects for therapy typicallycomprise two treatment groups that can be distinguished by clinicalcriteria. Subjects with “advanced disease” or “high tumor burden” arethose who bear a clinically measurable tumor. A clinically measurabletumor is one that can be detected on the basis of tumor mass (e.g., bypalpation, CAT scan, sonogram, mammogram or X-ray; positive biochemicalor histopathologic markers on their own are insufficient to identifythis population). A pharmaceutical composition embodied in thisinvention is administered to these subjects to elicit an anti-tumorresponse, with the objective of palliating their condition. Ideally,reduction in tumor mass occurs as a result, but any clinical improvementconstitutes a benefit. Clinical improvement includes decreased risk orrate of progression or reduction in pathological consequences of thetumor.

A second group of suitable subjects is known in the art as the “adjuvantgroup.” These are individuals who have had a history of neoplasia, buthave been responsive to another mode of therapy. The prior therapy canhave included, but is not restricted to, surgical resection,radiotherapy, and traditional chemotherapy. As a result, theseindividuals have no clinically measurable tumor. However, they aresuspected of being at risk for progression of the disease, either nearthe original tumor site, or by metastases. This group can be furthersubdivided into high-risk and low-risk individuals. The subdivision ismade on the basis of features observed before or after the initialtreatment. These features are known in the clinical arts, and aresuitably defined for each different neoplasia. Features typical ofhigh-risk subgroups are those in which the tumor has invaded neighboringtissues, or who show involvement of lymph nodes.

Another group have a genetic predisposition to neoplasia but have notyet evidenced clinical signs of neoplasia. For instance, women testingpositive for a genetic mutation associated with breast cancer, but stillof childbearing age, can wish to receive one or more of theantigen-binding fragments described herein in treatment prophylacticallyto prevent the occurrence of neoplasia until it is suitable to performpreventive surgery.

Human neoplasia subjects having any of the following neoplasias:glioblastoma, melanoma, neuroblastoma, adenocarcinoma, glioma, softtissue sarcoma, and various carcinomas (including prostate and smallcell lung cancer) are especially appropriate subjects. Suitablecarcinomas further include any known in the field of oncology,including, but not limited to, astrocytoma, fibrosarcoma, myxosarcoma,liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitiveneural ectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma,pancreatic ductal adenocarcinoma, small and large cell lungadenocarcinomas, chordoma, angiosarcoma, endotheliosarcoma, squamouscell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinoma, andliver metastases thereof, lymphangiosarcoma,lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovioma,mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basalcell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceousgland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,testicular tumor, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma,Waldenstrom's macroglobulinemia, and heavy chain disease, breast tumorssuch as ductal and lobular adenocarcinoma, squamous and adenocarcinomasof the uterine cervix, uterine and ovarian epithelial carcinomas,prostatic adenocarcinomas, transitional squamous cell carcinoma of thebladder, B and T cell lymphomas (nodular and diffuse) plasmacytoma,acute and chronic leukemias, malignant melanoma, soft tissue sarcomasand leiomyosarcomas.

The subjects can have an advanced form of disease, in which case thetreatment objective can include mitigation or reversal of diseaseprogression, and/or amelioration of side effects. The subjects can havea history of the condition, for which they have already been treated, inwhich case the therapeutic objective will typically include a decreaseor delay in the risk of recurrence.

Accordingly, the invention provides a method of treating or preventing aneoplasia in a subject, the method comprising administering an effectiveamount of an immunoresponsive cell comprising a receptor that binds atumor antigen and activates the immunoresponsive cell (e.g., TCR, CAR)and a vector encoding a single-chain variable fragment (scFv) that bindsan antigen having immunosuppressive activity (e.g., CD47, PD-1, CTLA-4,and ligands thereof). In one embodiment, the neoplasia is selected fromthe group consisting of blood cancers (e.g. leukemias, lymphomas, andmyelomas), ovarian cancer, prostate cancer, breast cancer, bladdercancer, brain cancer, colon cancer, intestinal cancer, liver cancer,lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomachcancer, glioblastoma, and throat cancer. In another embodiment, thetumor antigen is one or more of carbonic anhydrase IX (CA1X),carcinoembryonic antigen (CEA), CDS, CD7, CDIO, CD19, CD20, CD22, CD30,CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, anantigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surfaceantigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40(EGP-40), epithelial cell adhesion molecule (EpCAM), receptortyrosine-protein kinases erb-B2,3,4, folate-binding protein (FBP), fetalacetylcholine receptor (AChR), folate receptor-α, Ganglioside G2 (GD2),Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2),human telomerase reverse transcriptase (hTERT), Interleukin-13 receptorsubunit alpha-2 (IL-13Ra2), light chain, kinase insert domain receptor(KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanomaantigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1),Mesothelin (MSLN), NKG2D ligands, cancer-testis antigen NY-ESO-1,oncofetal antigen (h5T4), prostate stem cell antigen (PSCA),prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), or Wilmstumor protein (WT-1).

As a consequence of surface expression of a receptor that binds a tumorantigen and activates the immunoresponsive cell (e.g., TCR, CAR) and avector encoding a single-chain variable fragment (scFv) that binds anantigen having immunosuppressive activity (e.g., CD47, PD-1, CTLA-4, andligands thereof), adoptively transferred human Tor NK cells are endowedwith augmented and selective cytolytic activity at the tumor site.Furthermore, subsequent to their localization to tumor or viralinfection and their proliferation, co-stimulatory ligand-expressing Tcells turn the tumor or viral infection site into a highly conductiveenvironment for a wide range of immune cells involved in thephysiological anti-tumor or antiviral response (tumor infiltratinglymphocytes, NK-, NKT-cells, dendritic cells, and macrophages).

In other embodiments, the invention provides methods for treatingsubjects with a pathogen infection (e.g., viral infection, bacterialinfection, fungal infection, parasite infection, or protozoalinfection). The invention is particularly useful for enhancing an immuneresponse in an immunocompromised subject. Exemplary viral infectionssusceptible to treatment using a method of the invention include, butare not limited to, Cytomegalovirus (CMV), Epstein Barr Virus (EBV),Human Immunodeficiency Virus (HIV), and influenza virus infections.

Accordingly, the invention provides a method of treating or preventing apathogen infection in a subject, the method comprising administering aneffective amount of an immunoresponsive cell as described herein.

Kits

The invention provides kits for the treatment or prevention of aneoplasia, pathogen infection, immune disorder or allogeneic transplant.In one embodiment, the kit includes a therapeutic or prophylacticcomposition containing an effective amount of an immunoresponsive cellcomprising an activating antigen receptor and a single-chain variablefragment (scFv) that binds an antigen having immunosuppressive activityin unit dosage form. In particular embodiments, the cells furthercomprise a co-stimulatory ligand. In some embodiments, the kit comprisesa sterile container which contains a therapeutic or prophylacticvaccine; such containers can be boxes, ampules, bottles, vials, tubes,bags, pouches, blister-packs, or other suitable container forms known inthe art. Such containers can be made of plastic, glass, laminated paper,metal foil, or other materials suitable for holding medicaments.

If desired the immunoresponsive cell is provided together withinstructions for administering the cell to a subject having or at riskof developing a neoplasia, pathogen infection, immune disorder orallogeneic transplant. The instructions will generally includeinformation about the use of the composition for the treatment orprevention of neoplasia, pathogen infection, immune disorder orallogeneic transplant. In other embodiments, the instructions include atleast one of the following: description of the therapeutic agent; dosageschedule and administration for treatment or prevention of a neoplasia,pathogen infection, immune disorder or allogeneic transplant or symptomsthereof; precautions; warnings; indications; counter-indications;overdosage information; adverse reactions; animal pharmacology; clinicalstudies; and/or references. The instructions may be printed directly onthe container (when present), or as a label applied to the container, oras a separate sheet, pamphlet, card, or folder supplied in or with thecontainer.

EXAMPLES

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

Example 1. T Cells Co-Expressing a Chimeric Antigen Receptor (CAR) andan Anti-SCD47 scFv Eradicated Tumors

An scFv that specifically binds human CD47 was generated and humanperipheral blood T cells modified with this scFv and a CAR recognizing atumor antigen (CD19), demonstrated in vitro anti-tumor efficacy as wellas enhanced anti-tumor efficacy in a preclinical model.

Constructs comprising 1928z-2A-B6H12.2 (FIGS. 1-5) were generated asconfirmed by sequencing the CAR and scFv sequences. In addition, controlconstructs were generated with a CAR specific for the ovarian cancerantigen, MUC-CD, termed 4H1128z (FIG. 6). Stable producer cell lineswere generated for the constructs utilizing the kappa leader sequence,and verified by flow cytometry (FIG. 7A). Supernatant from the packagingcell lines, containing secreted anti-CD47 scFV was able to block CD47antibody from binding to Nalm-6 and Raji tumor cells in a flow cytometrybased assay. Tumor cells incubated with supernatant from the packagingcells were also stained with an anti-c-myc tag antibody, to demonstratebinding of the scFv (FIG. 7B).

The packaging cells were utilized to transduce human peripheral blood Tcells where transduction efficiency was assessed by flow cytometricanalysis of CAR expression (FIG. 8A). The secreted scFv was able tofunction in an autocrine fashion, where an anti-CD47 antibody hasreduced binding to 1928z-2A-B6H12.2 T cells compared to 1928z T cells.Positive staining with anti-c-myc tag antibody indicated bound scFv(FIG. 8B). The phenotype of the transduced T cells was investigated byflow cytometry and demonstrated to be similar between 1928z-2A-B6H12.2and 1928z T cells, with the exception of CD62L, which was found to bedecreased on 1928z-2A-B6H12.2 T cells (FIG. 9A). The function of T cellsproducing the anti-CD47 scFv was investigated using multiparameter flowcytometry and a standard 51Cr release assay. It was demonstrated that1928z-2A-B6H12.2 T cells have equivalent cytokine production andcytotoxic function when compared to 1928z T cells (FIGS. 9B and 9C).

The ability of 1928z-2A-B6H12.2 T cells to respond to tumor in vivo wasinvestigated using a preclinical SCID-Beige mouse model. SCID-Beige micewere injected intravenously with 1×10⁶ Nalm-6 tumor cells modified toexpress Firefly luciferase, 3 days later mice were treated with 5.7×10⁶CAR⁺ 1928z or 1928z-2A-B6H12.2 or control4H1128z-2A-B6H12.2 T cells,also injected intravenously. Tumor progression was monitored clinicallyand with bioluminescent imaging. Treatment of tumor bearing mice with1928z-2A-B6H12.2 T cells reduced tumor burden and enhanced the survivalof tumor bearing mice compared to treatment with 1928z T cells (FIGS.10A and 10B).

Example 2. T Cells Co-Expressing a Chimeric Antigen Receptor (CAR) andan Anti-Human PD-1 scFv had Increased Proliferation and RetainedExpression of CAR

An anti-human PD-1 scFv was generated based on the V_(H) and V_(L)chains from an anti-PD-1 antibody (clone 5C4) (U.S. Pat. No. 8,008,449).The 5C4 scFv was designed to include the kappa leader sequence, a serineglycine linker and the c-myc tag (FIG. 11). This scFv construct wascloned into SFG retriviral backbone to generate 1928z-2A-5C4 and4H1128z-2A-5C4 (FIGS. 12 and 13). To develop a high affinity scFv thatbinds to human PD-1 (e.g., for expression in a 1928z/4H1128z CART cell),a human antibody phage display library is screened to determine scFvsthat specifically bind human PD-1 (and potentially mouse PD-1).

Stable 293G1v9 packaging cell lines were produced and expression of the1928z CAR and 4H1128z CAR was assessed by flow cytometry (FIG. 14).Supernatant from these packaging cells was utilized to transduce humanperipheral blood T cells and transduction efficiency was assessed byflow cytometry to detect CAR expression (FIG. 15).

The ability of this anti-human PD-1 scFv to increase proliferation of Tcells in response to artificial antigen presenting cells (aAPCs) wasinvestigated. PD-L1 positive tumor cells and 3T3 aAPCs were generatedfor the study (FIG. 16). Following co-culture of transduced T cells with3T3 aAPCs expressing human CD19, human B7.1 and human PD-L1, 1928z-2A-C4T cells had increased proliferation and retained expression of CARcompared to 1928z T cells (FIG. 17). The phenotype and anti-tumorfunction of T cells co-expressing 1928z CAR and anti-PD1 scFV can bedetermined using flow cytometry, luminez cytokine analysis studies,⁵¹Chromium release assays, and SCID-Beige preclinical model to determinein vivo anti-tumor function.

Example 3. Co-Expression of a Chimeric Antigen Receptor (CAR) and anAnti-Mouse PD-1 scFv Stimulates Mouse T Cells

An anti-mouse PD-1 scFv was generated based on the V H and VL from theanti-PD-1 antibody clone J43 (U.S. Pat. No. 7,858,746 to Honjo et al.).The J43 scFv was designed to include the mouse kappa chain leadersequence, a serine glycine linker and the c-myc tag (FIG. 18). This scFvconstruct was cloned into SFG retroviral backbone expressing the CARtargeting human CD19 or human MUC-CD that signals through mouse C28 andmouse CD3zeta, therefore stimulating mouse T cells. These constructs19m28mz-IRES-J43 (FIG. 19) and 4H11m28mz-IRES-J43 (FIG. 20) are used togenerate stable Phoenix packaging cells lines and genetically modifyprimary murine T cells, as previously described (Lee et al., Cancer Res2011, 71(8):2871). Mouse 19m28mz and 19m28mz-IRES-J43 T cells arecultured with EL4 thymoma tumor cells that have been modified to expresshuman CD19 and mouse PD-L1, proliferation of mouse T cells to monitorviable cell counts and CFSE labeling.

For murine T cells expressing the 4H11m28mz CAR that target the MUC-CDantigen, function can be assessed in response to IDS tumor cellsmodified to express MUC-CD and mouse PD-L1 (Chekmasova et al., ClinCancer Res, 2010, 16:3594). A human scFv that binds murine PD-1, asdescribed above, is cloned into the SFG-19m28mz and 4H11m28mz vectorconstructs and used to modify murine T cells. Syngeneic models to assessthe in vivo anti-tumor effects of the T cells modified to express humanscFv that binds murine PD-1 are available: an ovarian cancer tumor modelutilizing ID8-MUC-CD tumor cells, which are inoculated intraperitoneallyinto C57BL/6 mice; and transgenic mice that express human CD19 in placeof mouse CD19, which are inoculated with EL4 thymoma tumor cellsmodified to express human CD19 (Pegram et al., Blood 2012,119(18):4133). Thus, the anti-tumor effect can be evaluated in animmune-competent model, therefore, allowing assessment of the impact ofthe anti-PD-1 scFv on the tumor microenvironment.

Example 4. Co-Expression of a Chimeric Antigen Receptor (CAR) andAgonistic scFv in Immune Cells

In one embodiment, the invention provides an immune cell that expressesan antigen binding receptor (e.g., CAR or TCR) and a single-chainvariable fragment (scFv) that binds an antigen having agonisticimmunostimulatory activity (e.g., CD28, OX-40, 4-1BB, and ligandsthereof). To generate agonistic scFvs targeting costimulatory molecule4-1BB, the 3H3 hybridoma cell line was obtained (Shuford et al., J ExpMed 1997, 186:47-55; provided by Professor Mick Croft (La JollaInstitute for Allergy and Immunology)). To generate agonistic scFvstargeting costimulatory molecule OX-40, OX-86 hybridoma cell line wasobtained (al-Shamkhani et al., Eur J Immunol1996, 26(8):1695-9; EuropeanCollection of Cell Cultures (Catalogue number 96110601)). Hybridoma mRNAwas isolated from cells using a QIAgen RNAeasy kit, as permanufacturer's instruction (QIAgen, CA, USA), and cDNA was then preparedusing New England Biolabs Protoscript AMV First strand cDNA synthesiskit, as per the manufacturers instruction (New England Biolabs, MA,USA). The variable heavy (VH) and light (VL) chains were then PCRamplified using the following degenerate primers:

Orlandi Primers (Orlandi et al.,Proc. Natl. Acad. Sci. 1989, 86:3833-37) VHFOR: [SEQ ID NO: 28]5′-tga gga gac ggt gac cgt ggt ccc ttg gcc cca g-3′ VH1BACK:[SEQ ID NO: 29] 5′-agg tsm arc tgc ags agt cwg g-3′ VKFOR:[SEQ ID NO: 30] 5′-gtt aga tct cca gcttgg tcc c-3′ VK1BACK:[SEQ ID NO: 31] 5′-gac att cag ctg acc cag tct cca-3′ Cooper Primers(Wang et al., Blood 2002, 99:2459-2467) Vk [SEQ ID NO: 32]5′-GGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC-3′, Ck [SEQ ID NO: 33]5′-CTCATTCCTGTTGAAGCTCTTGACAATGGG-3′; RACE PRIMERS(Kettleborough et al., Eur. J Immunol1993, 23:206-211) VKfr1a:[SEQ ID NO: 34] Ata tcc atg gca gac gtc cag atg atc cag tct cca Vkfr1b:[SEQ ID NO: 35] ata tcc atg gca gac att gtg ctg act cag tct cc Vkfr1c:[SEQ ID NO: 36] ata tcc atg gca gat gtt gtg atg acc caa act cca Vkfr1d:[SEQ ID NO: 37] ata tcc atg gca caa att gtt ctc acc cag tct cc Vkfr1e:[SEQ ID NO: 38] ata tcc atg gca gac att gtg atg aca cag tct cca Vkfr1f:[SEQ ID NO: 39] ata tcc atg gca gat att gtg atg acg cag gct gca Vkfr1g:[SEQ ID NO: 40] ata tcc atg gca gac att gtg atg acc cag tct cReverse Kappa: [SEQ ID NO: 41] gct tca aca gga atg agtgtt aac tcg agg tag

To assemble the VH and VL into a scFv, a serine glycine linker is addedduring PCR of the VH And VL chains, as well as c-myc tag and murine IgKappa chain or CDS leader sequence. The resulting polynucleotide iscloned into an existing retroviral expression vector (SFG backbone)encoding the 1928z chimeric antigen receptor (CAR), to generateSFG-1928z-2A-3H3 or 1928z-2A-OX86.

Stable packaging cell lines are generated as described for theanti-mouse PD-1 J43 scFv, and tested in a similar murine model ofadoptive T cell transfer.

The results presented herein indicate genetically modified CAR T cellsexpressing scFv molecules (“armored CART cells”) are immunoresponsiveand can overcome “hostile” tumor microenvironment, and, thus, areeffective in the treatment of neoplasia. CAR⁺ T cells are modified tosecrete antagonistic scFvs with immune regulatory functions (FIG. 21).Upon activation of the CAR to cognate antigen (1), armored CAR modifiedT cells may be induced to secrete scFvs antagonistic to the inhibitoryPD-1 T cell receptor on both infused CAR modified T cells and endogenousanti-tumor T cells enhancing anti-tumor effector function (2), inducedto secrete scFvs antagonistic to the inhibitory CTLA-4 T cell receptoron both infused CAR modified T cells and endogenous anti-tumor T cellsenhancing anti-tumor effector function (3), or induced to secrete anscFv antagonistic to the CD47 receptor expressed on the tumor cellreversing the cloaking the tumor cell from recognition by the hostinnate anti-tumor immune response leading to recognition and eradicationof tumor by host macrophages.

Results reported herein were obtained using the following methods andmaterials unless indicated otherwise.

Generation of Anti-CD47 B6H12.2 scFv

The B6H12.2 hybridoma cell line was obtained from the American TissueCulture Collection (ATCC, VA, USA; catlogue number HB-9771). B6H12.2mRNA was isolated from hybridoma cells using a QIAgen RNAeasy kit,according to manufacturer's instruction (QIAgen, CA, USA), and cDNA wasprepared using New England Biolabs Protoscript AMV First strand cDNAsynthesis kit, according to manufacturer's instruction (New EnglandBiolabs, MA, USA). The variable heavy (VH) and light (VL) chains werePCR amplified using primers designed to incorporate the Kappa leadersequence, serine glycine linker and c-myc tag (see FIG. 1) as follows:

Primer 1. B6Hl2.2 VH forward primer [SEQ ID NO: 42]:5′-CCA TGG AGA CAG ACA CAC TCC TGC TAT GGG TAC TGC TGC TCT GGGTTC CAG GTT CCA CTG GTG ACG AGG TGC TGC AGC TGG TGG AGT CCG GGG-3′Primer 2. B6Hl2.2 VH reverse primer [SEQ ID NO: 43]:5′-AGA TCC ACC TCC ACC AGA TCC ACC TCC ACC TGA TCC ACC TCC ACCTGA GGA GAC GGT GAC TGA GGT TCC TTG ACC-3′ Primer 3. B6Hl2.2 VL forwardprimer [SEQ ID NO: 44]: 5′-GGT GGA GGT GGA TCA GGT GGAGGT GGA TCT GGT GGA GGT GGA TCT GAC ATT GTG ATG ACT CAG TCT CCAGCC ACC-3′ Primer 4. B6Hl2.2 VL reverse primer [SEQ ID NO: 45]:5′-CTC GAG TTA CAG ATC CTC TTC TGA GAT GAG TTT TTG TTG TTT GATTTC CAG CTT GGT GCC TCC ACC GAA CG-3′

In addition to the above design, an scFv with the CD8L sequence wasgenerated to determine an efficient leader sequence for exportation ofthe scFv from T cells using the following alternative forward primer:

Primer 5. B6H12.2 VH CD8L forward [SEQ ID NO: 46]:5′-TAT ACC ATG GCC TTA CCA GTG ACC GCC TTG CTC CTG CCG CTG GCCTTG CTG CTC CAC GCC GCC AGG CCG GAG GTG CAG CTG GTG GAG TCC GGG-3′

The VH and VL PCR products were cloned into pCR2.1TOPO, according tomanufacturer's instruction (Invitrogen, NY, USA). Sequencing using M13F2and M13R2 primers (Invitrogen) was performed by the MSKCC DNA sequencingcore facility to confirm the sequence of both the VH and VL products.Overlapping PCR was performed using the VH and VL PCR products andprimers 1 or 5 and 4 to generate the anti-CD47 scFv (see FIG. 1).

The anti-CD47 scFv construct was cloned into an existing retroviralexpression vector (SFG backbone) encoding the 1928z chimeric antigenreceptor (CAR), to generate SFG-1928z-2A-B6H12.2. TheSFG-1928z-2A-B6H12.2 DNA was sequenced to confirm the sequence.

Generation of Stable Packaging Cell Line for Human T Cells

To generate stable packaging cell lines, H29 cells were transientlytransfected with 10 μg of SFG-1928z-2A-B6H12.2 DNA using a Promegacalcium phosphate transfection kit, according to manufacturer'sinstructions (Promega). Supernatant from H29 supernatant was used totransduce 293G1v9 cells, which were subsequently sub-cloned to generatestable packaging cells. Selection of two sub-clones (clone 5 and clone6) was based upon expression of both 1928z CAR and ability of 293G1v9supernatant to transduce human peripheral blood T cells (as determinedby flow cytometry following staining with 12dll antibody). Transductionof human peripheral blood T cells was performed as described previously(Brentjens et al., Clin Cancer Res 2007, 13(18Pt1):5426).

Assessment of Anti-CD47 scFv Production/Function

Production of anti-CD47 scFv from 1928z-2A-B6H12.2 293G1v9 andtransduced human peripheral blood T cells was determined by incubatingCD47⁺ tumor cells (Raji and Nalm-6) in supernatant from these cells.Tumor cells were subsequently washed and stained with fluorescentlyconjugated anti-c-myc tag antibody (Cell Signaling, MA, USA) to detectsupernatant derived protein bound to the tumor cells. Tumor cells werealso stained with fluorescently conjugated anti-CD47 (clone B6H12.2,eBioscience) to detect ability of B6H12 scFv to block CD47.

In Vivo Adoptive Transfer Model

Mice were injected intravenously with 1×106 Nalm-6 modified to expressFirefly luciferase (day 0). On day 3, mice were treated with 5.7×106CAR⁺ T cells, also inoculated intravenously. Tumor progression wasmonitored clinically and using bioluminescent imaging, as describedpreviously (Santos et al., Nature Medicine 2009, 15(3):338).

Generation of 5C4 Anti-Human PD-1 scFv

The sequence for an antibody that specifically binds human PD-1, clone5C4, was obtained, as described above. This sequence was modified toinclude a kappa leader sequence, serine glycine linker and the c-myc tagand purchased from GeneArt (Invitrogen, FIG. 9). Cloning of this scFvinto SFG retroviral backbone, generation of stable packaging cells,transduction of human peripheral blood T cells and assessment oftransduction efficiency was achieved as described above.

Assessment of Anti-Human PD-1 Function

The PD-1ligand, PD-L1 was PCR amplified from SKOV3 (ATCC) tumor cellsthat were incubated in 200 ng/ml recombinant human Interferon-gamma (RnDsystems, MN, USA). Primers used to amplify human PD-L1 are shown below:

Primer 6. Human PD-L1 forward primer [SEQ ID NO: 47]5′-CACGTGCCATGGATGAGGATAT TTGCTGTCTT TATAT-3′Primer 7. Human PD-L1 reverse primer [SEQ ID NO: 48]5′CTCGAGTTACGTCTCCTCCAAATGTGTATCACTTT3′

The human PD-L1 sequence was cloned into a SFG retroviral backbone, andtransduced into 3T3, Raji and Nalm-6 cell lines as described previously(Brentjens et al., Clin Cancer Res 2007, 13(18 Pt 1):5426). Cells werestained with anti-PD-L1 (clone MIH1, BD Pharmingen, CA, USA) and FACSsorted to ensure the total cell population expressed PD-L1 (FIG. 14).

Human 1928z-2A-5C4 and 1928z T cells were cultured with 3T3(CD19/B7.1/PD-L1) aAPCs and viable cell counts were performed utilizingtrypan blue exclusion and flow cytometry was performed to determineexpression of the CAR. This was correlated to expansion of T cells whencultured with 3T3 (CD19/B7.1) aAPCs.

Generation of Anti-Mouse PD-1 scFv

The sequence for an antibody that specifically binds murine PD-1, clonename J43, was obtained, as described above. This sequence was modifiedto include a Kappa chain leader sequence and c-myc tag sequence, with aserine glycine linker to form a scFv and purchase from GeneArt(Invitrogen, FIG. 16). This was cloned into an existing retroviralexpression vector (SFG) encoding a murine CAR, where signaling ismediated through mouse CD28 and CD3 zeta molecules. The 19m28mz-IRES-J43and 4H11m28mz-IRES-J43 were generated to target B cell and ovarian tumorrespectively (FIGS. 17 and 18).

Assessment of Anti-Mouse PD-1 Function

The PD-1ligand, PD-L1 was PCR amplified from Renca tumor cells (ATCC),primers used to amplify mouse PD-L1 are shown below:

Primer 8. Mouse PD-L1 forward primer [SEQ ID NO: 49]5′-TAT TAC ACG TGT TAC ATG AGG ATA TTT GCT GTC TTT-3′Primer 9. Mouse PD-L1 reverse primer [SEQ ID NO: 50]5′TAT AGG ATC CTC GAG GAT GTT ACG TCT CCT CCA AAT GTG TA 3′

The anti-mouse PD-1 scFv was cloned into an SFG retroviral backbone, andtransduced into 3T3 aAPCs, IDS and EL4 cell lines. Cells stained withanti-PD-L1 (clone MIH1 BD Pharmingen) are FACS sorted to ensure thetotal cell population expressed PD-L1.

CTL Chromium Release Killing Assays

Target cells expressing desired antigen were labeled with 51Cr andco-cultured with T cells at decreasing effector: target ratio's. After 4hours of culture, supernatant was removed and used to measureradioactivity released from chromium. Specific lysis was determined bysubtracting background radioactivity of target cells not cultured with25T cells and dividing by the radioactivity measured from target cellscompletely lysed by using 0.2% Triton X-100.

Example 5. Blocking CD47 Improves CAR T Cell Therapy

T cells can be genetically modified to target tumor antigens through theexpression of a chimeric antigen receptor (CAR). Adoptive transfer ofCD19-specific CAR T cells has shown clinical efficacy in some patientswith hematological malignancies, however chronic lymphocytic leukemiapatients with bulky lymphadenopathy have suboptimal responses to CAR Tcell therapy. Furthermore, CAR T cell therapy has failed to demonstrateefficacy against solid tumors in clinical trials. To enhance theclinical efficacy of CAR T cells we propose to recruit an innateanti-tumor immune response through the secretion of a CD47-blockingsingle chain variable fragment (scFv) from CAR T cells. Previous studiesshow that blocking the interaction between CD47 on tumor cells and SIRPaon macrophages results in phagocytosis of tumor cells. To harness thiseffect, T cells were modified to express the CD19-specific CAR (1928z)and secrete a scFv specific for human CD47, cloned from the B6H12.2hybridoma (1928z/B6H12.2 T cells). 1928z/B6H12.2 T cells were shown tosecrete a functional scFv specific for human CD47, which did not affectCAR-mediated cytokine secretion or cytotoxicity in vitro. Supernatantfrom 1928z/B6H12.2 T cells but not 1928z T cells stimulated macrophagesto phagocytose tumor cells in vitro. Adoptive transfer of 1928z/B6H12.2T cells mediated enhanced anti-tumor effects and eradicated Nalm6 tumorsin a preclinical murine model. This novel strategy combines CAR Tcell-mediated effects and innate immune cell-mediated destruction oftumor cells, which may improve the anti-tumor efficacy of CAR T celltherapy

Example 6. Enhancing Anti-Tumor Efficacy of Chimeric Antigen ReceptorModified T-Cells Through Constitutive CD40L Expression

Adoptive cell therapy with genetically modified T-cells expressing achimeric antigen receptor (CAR) is a promising therapy for patients withB-ALL. However, in most clinical trials CAR-modified T-cells have failedto demonstrate a significant therapeutic benefit, specifically in thecontext of low grade B-cell malignancies and solid tumors. In theexperiments presented in this example section, we further enhance theanti-tumor efficacy of CAR-modified T-cells by engineering T-cells toconstitutively express CD40 ligand (CD40L, CD154). T-cells modified toconstitutively express CD40L (CD40L-modified T-cells) increasedproliferation and secretion of pro-inflammatory TH1 cytokines. Further,CD40L-modified T-cells augmented the immunogenicity of CD40+ tumor cellsby the upregulation of co-stimulatory molecules (CD80 and CD86),adhesion molecules (CD54, CD58, and CD70), HLA molecules (Class I andHLA-DR) and the Fas death receptor (CD95) on the surface of the tumorcell. Additionally, CD40L-modified T-cells induced maturation andstimulated secretion of the pro-inflammatory cytokine IL-12 by monocytederived dendritic cells. Finally, tumor targeted CAR/CD40L T-cellsincreased cytotoxicity against CD40+ tumors and extended the survival oftumor bearing mice in a xenotransplant model of systemic lymphoma. Thesepre-clinical data support the clinical application of CAR T-cellsadditionally modified to constitutively express CD40L with anticipatedenhanced anti-tumor efficacy and improved clinical outcome.

Materials and Methods.

Cell Culture

DoHH2, Raji, and NALM-6 (American Type Culture Collection) tumor celllines were maintained in RPMI 1640 medium (Gibco) supplemented with 10%heat inactivated fetal bovine serum (FBS), nonessential amino acids,sodium pyruvate, HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonicacid) buffer, and 2-Mercaptoethanol (Invitrogen). The 293GP-GLV9retroviral producer cell lines have been described previously and werecultured in DMEM (Invitrogen) supplemented with 10% FBS.²⁹ NIH-3T3artificial antigen-presenting cells (AAPC) were cultured in DMEMsupplemented with 10% heat-inactivated donor calf serum (DCS) asdescribed previously.³⁰ Human T-cells were isolated from peripheralblood of healthy donors under Memorial Sloan-Kettering Cancer Center(MSKCC) IRB-approved protocol 95-054 using BD Vacutainer CPT tubes(Becton Dickinson) as per the manufacturer's instructions. PatientT-cell and CLL cells were obtained from patients undergoing treatmentunder MSKCC IRB-approved protocol 06-138 and isolated using DynabeadsClinExVivo CD3/CD28 beads (Invitrogen). T-cells were cultured in RPMI1640 supplemented with 10% FBS and 20 IU/mL IL-2 (R&D Systems). Monocytederived dendritic cells (moDCs) were obtained from tissue cultureplastic-adherent peripheral blood mononuclear cells (PBMCs) of healthydonors and cultured in RPMI 1640 supplemented with 1% pooled human ABserum, HEPES buffer, 2-Mercaptoethanol (Invitrogen), interlukin-4 (IL-4;500 IU/ml—R&D Systems) and granulocyte-monocyte colony-stimulatingfactor (GM-CSF; 1000 IU/ml—R&D Systems) as previously described.³¹ Allmedia were supplemented with 2 mmol/L L-glutamine (Invitrogen), 100units/mL penicillin, and 100 μg/ml streptomycin (Invitrogen)

Construction of Retroviral Constructs

Human CD40L cDNA was PCR amplified from isolated healthy donor PBMCsusing the following primers (1)5′-CACGTGCATGATCGAAACATACAACCAAACTTCTCCCCGATCTGC-′3 [SEQ ID NO: 3] and(2) 5′-CTCGAGGGATCCTCAGAGTTTGAGTAAGCCAAAGGA-3′ [SEQ ID NO:4] (FIG. 22A).A gamma-retroviral vector encoding human CD40L was constructed using theSFG vector backbone.³² Construction of 1928z and Pz1 (anti-prostatespecific membrane antigen CAR; anti-PSMA) SFG-vector has been previouslydescribed.³³′³⁴ Construction of 1928z-IRES-40L and Pz1-IRES-40Lgamma-retroviral vector was generated using overlapping PCR (FIG.26A).³⁵

Retroviral Transduction of Human T-Lymphocytes

Generation of stable 293GP-GLV9 retroviral producer cell lines andgenetic modification of human T-cells has been previouslydescribed.^(29,36) For T-cell transduction isolated healthy donor PBMCswere activated with phytohemagglutinin (PHA) at 2 μg/mL (Sigma), whereaspatient derived T-cells were isolated, activated, and expanded usingDynabeads ClinExVivo CD3/CD28 beads following the manufacturer'srecommendations. Activated T-cells were retrovirally transduced onretronectin-coated non-tissue culture treated plates as previouslydescribed.³⁶ Gene transfer was assessed on day 7 by flow cytometry.Control mock-transduced T-cells were generated in the same manner exceptsupernatant was derived from empty 293GP-GLV9 cell cultures.Proliferation of CD40L-modified T-cells was assessed by the Guava®easyCyte™ cell counter with Guava® ViaCount reagent (EMD Millipore) asper manufacturer's instructions. Expansion of modified T-cells for invivo experiments was performed using AAPCs derived from NIH-3T3 murinefibroblast genetically engineered to express the target antigen (CD19 orPSMA) along with co-stimulation (CD80) as previously described.³⁰

Co-Culture Assays

Tumor cells (DOHH2, Raji, Ph⁺ ALL 3.1, NALM-6) were co-cultured at aratio of 5:1 with CD40L-modified T-cells and mock-transduced T-cells.Flow cytometry was performed after three days to determine phenotype oftumor cells. moDCs (2.5×10⁵) were co-cultured with autologousCD40L-modified T-cells or mock-transduced T-cells at a 1:5 ratio andtissue culture supernatant was analyzed after 24 hours for IL-12p70 on aLuminex IS100 system (see below). moDCs were also co-cultured at a ratioof 5:1 with CD40L-modified T-cells and mock-transduced T-cells andphenotype of moDC was analyzed by flow cytometry 24 hours later.

Cytotoxicity Assay

The cytolytic capacity of transduced T-cells was determined usingstandard ⁵¹Cr release assay as previously described.³⁴

Cytokine Detection Assays

Cytokine detection in tissue culture supernatant was assessed using theMILLIPLEX Human Cytokine Detection System (Millipore Corp.) inconjunction with the Luminex IS100 system and IS 2.3 software (LuminexCorp.) as per manufacturer's instructions.

Flow Cytometry

Flow cytometry was performed using a FACScan cytometer and data analyzedusing FlowJo version 9.2 software (Tree Star). CAR expression wasdetected using CAR specific Armenian hamster monoclonal antibody 19E3(1928z) and 12D11 (1928z and Pz1, MSKCC monoclonal antibody facility).CD40L expression was detected using mouse anti-human CD154 (BDBiosciences). Human T-cells were stained with mouse anti-human CD3 (BDBiosciences), CD4, and CD8 (Invitrogen). moDCs were stained using mouseanti-human CD11b, HLA-DR, CD83, and CD86 (Invitrogen). DOHH2, Raji, andNALM6 tumor cell phenotype was detected using mouse anti-human CD19,CD40, CD54, CD80 CD86, HLA-Class I and HLA-DR (Invitrogen), CD58, CD70,and CD95 (BD Biosciences).

CAR T-Cell In Vivo Studies

We inoculated 8 to 12 week-old SCID/Beige(CB17.Cg-Prkdc^(scid)Lyst^(bg-J)/Crl) mice (Charles River Laboratories)with DOHH2 tumor cells (5×10⁵ cells) by intravenous injection. Two dayslater mice were infused intravenously with transduced T-cells (1×10⁷ CART-cells). Tumor progression was monitored clinically and mice wereeuthanized when disease became clinically evident (development of hindlimb paralysis or decreased response to stimuli). All murine studieswere done in accordance with a Memorial Sloan-Kettering Cancer CenterInstitutional Animal Care and Use Committee approved protocol(00-05-065).

Statistical Analysis

All analyses were calculated using Graphpad Prism 5.0 software, survivaldata were assessed using a log-rank analysis and all other analyses wereachieved with a Mann-Whitney test (one-tailed).

Results

Constitutive Expression of CD40L by Human T-Cells

We initially transduced T-cells from healthy donor with a CD40Lretroviral vector (FIG. 22A). Retroviral transduction of T-cells withthe CD40L gene routinely resulted in >40% gene transfer with stableexpression of CD40L in both CD4+ and CD8+ T-cell subsets (FIG. 22B).Proliferation of CD40L-modified T-cells was significantly increasedcompared to mock-transduced T-cells generated from the same three donors(FIG. 22C). Tissue culture media from CD40L-modified T-cells wasanalyzed and shown to have significantly increased soluble CD40L(sCD40L) as expected, as well as significantly increased secretion ofthe pro-inflammatory cytokines IFN-γ and GM-CSF when compared to themock-transduced T-cells (FIG. 22D).

CD40L-Modified T-Cells Alter the Phenotype of Both CD40+ Tumor CellLines and Patient Derived CLL Cells

To investigate the ability of the CD40L/CD40 pathway to modify thephenotype of tumor cells a co-culture of CD40+ B-cell tumor cells andCD40L-modified T-cells or mock-transduced T-cells was performed.Cultures with CD40L-modified T-cells, but not mock-transduced T-cells,led to the upregulation of co-stimulatory molecules (CD80 and CD86),adhesion molecules (CD54, CD58, and CD70), HLA molecules (HLA Class Iand HLA-DR), and the Fas death receptor (CD95) on the surface of DOHH2tumor cells (FIG. 23A). Phenotypic changes are also evident when DOHH2tumor cells are cultured in conditioned media from CD40L-modified T cellwhich contains elevated levels of sCD40L (FIG. 28). To determine if CD40expression on the tumor cell is a requisite to alter tumor cellphenotype co-culture of the CD40-tumor cell line (NALM6) withCD40L-modified T-cells and mock-transduced T-cells was performed. Thesestudies resulted in no alteration in the phenotype demonstrating theneed for CD40 expression by the tumor to induce CD40L mediated changesin tumor cell phenotype (FIG. 23B).

To further verify this effect in a clinically relevant setting weco-cultured CD40L-modified T-cells derived from patients with CLL withautologous CLL tumor cells. Retroviral transduction of CLL patientderived T-cells routinely resulted in >40% gene transfer with stableexpression of the CD40L gene (FIG. 24A). In this setting patient derivedCD40L-modified T-cells, but not mock-transduced T-cells, demonstratedthe capacity to upregulate co-stimulatory molecules, adhesion molecules,HLA molecules and the Fas death receptor on the surface of theautologous CLL cells (FIG. 24B).

CD40L-Modified T-Cells Induce IL-12p70 Secretion and Mediate Maturationof moDCs

Given the role of CD40L in DC maturation and secretion of thepro-inflammatory cytokine IL-12 we next investigated if CD40L-modifiedT-cells could induce the same effect when co-cultured with autologousmoDCs. Significantly, we found CD40L-modified T-cell induced secretionof IL-12p70 in the co-cultures containing moDCs and autologousCD40L-modified T-cells from three separate donors (FIG. 25A). Maturationof moDCs as determined by upregulation of surface co-stimulatorymolecules (HLA-DR, CD86, and CD83) was also seen following co-culturewith CD40L-modified T-cells but not following co-culture withmock-transduced T-cells (FIG. 25B).

Expression of Both CAR and CD40L by T-Cells Results in Enhanced In Vitroand In Vivo Cytotoxicity

We next assessed the ability of T-cells to express both the anti-CD19CAR (1928z) and CD40L using a bi-cistronic retroviral vector(1928z/CD40L; FIG. 26A). Transduction of T-cells routinely resultedin >40% expression of both 1928z and CD40L (1928z/CD40L T-cells; FIG.26B). Control retroviral vectors were also generated including theanti-CD19 CAR (1928z) and anti-PSMA CAR (Pz1 and Pz1/CD40L; FIG. 26B).To assess in vitro anti-tumor activity of 1928z/CD40L T-cells, astandard 4 hour ⁵¹Cr release assay was performed. Constitutiveexpression of CD40L statistically enhanced the lytic capacity of 1928zT-cells against CD19+ tumor cells when compared to a panel of controlT-cells including T cells modified to express the 1928z CAR alone (FIG.26C). Enhanced cytotoxicity is also demonstrated against otherCD19+/CD40+ tumor cell lines (FIG. 29).

To investigate the in vivo antitumor activity of 1928z/CD40L T-cells weutilized a xenotransplant model of systemic DOHH2 lymphoma. We havepreviously observed that systemic DOHH2 tumor cells are markedlyrefractory to CD19-targeted CAR T-cell therapy in SCID/Beige mice. Toassess whether further modification of CAR T-cells with CD40L couldenhance the anti-tumor efficacy in this model we inoculated and treatedSCID/Beige mice bearing systemic DOHH2 tumor with CAR/CD40L T-cells.Significantly, treatment with 1928z/CD40L T-cells compared to treatmentwith 1928z T-cells or control T-cells (Pz1 and Pz1/CD40L T-cells)demonstrated enhanced survival and resulted in long-term survival in 30%of mice treated with 1928z/40L T-cells (FIG. 27).

Discussion

Adoptive therapy utilizing CAR T-cells has shown promising clinicalresponses in patients with B-cell malignancies.²⁻⁴ These studies havedemonstrated the potency of CAR T-cells as the sole anti-tumor effectorcell. However, this approach may have limited success against tumorswith a robust immunosuppressive tumor microenvironment.⁵ Furthermore, intheir current form CAR T-cells have not demonstrated the ability respondto tumor escape following target antigen loss.⁶ One possible method toovercome these limitations is to further engineer CAR T-cells throughthe constitutive expression of CD40L in effort to improve T-cellcytolytic capacity/proliferation, augment tumor immunogenicity, andimprove DC antigen presentation/function. Modification of CAR T-cellsthrough the constitutive expression of CD40L may also further activatean endogenous immune response thereby enhancing anti-tumor efficacy.

To assess the role of constitutive expression of CD40L by T-cells wefirst developed a retroviral vector containing the CD40L gene alone.When transduced in T-cells both constitutive expression of CD4⁺ and CD8⁺T-cell subsets are demonstrated (FIG. 22B). While more commonlyassociated with CD4⁺ T-cells, CD40L expression and helper function inmemory CD8⁺ T-cells has recently been reported.³⁷ CD40L expression isalso known to enhance T-cell proliferation and secretion ofpro-inflammatory TH1 cytokines (IFN-γ, GM-CSF).^(21,22) CD40L-modifiedT-cells demonstrate the ability to secrete pro-inflammatory cytokinesand enhanced proliferation as compared to similarly activated but mocktransduced T-cells from the same donor (FIGS. 22C and 22D). ArmingT-cells through the constitutive expression of CD40L has the potentialto enhance their anti-tumor function/activation.

The downregulation of cell surface proteins including HLA Class I,co-stimulatory molecules and/or adhesion molecules is often employed bytumors to avoid immune recognition.^(5,38,39) Apoptotic resistance canalso occur with the loss of the Fas death receptor on the surface ofmalignant cells.⁴⁰ To counteract this, CD40L can interact with CD40 onmalignant cells to mediate the up-regulation of co-stimulatory molecules(CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), HLAmolecules (HLA Class I and HLA-DR) and facilitate apoptosis through theFas/FasL pathway on malignant B-cell tumors.^(41,42) CD40L-modifiedT-cells modified the phenotype of CD40+ tumor cells resulting in theupregulation of these critical surface proteins thereby counteractingthe tumor cells' ability for immune evasion (FIG. 2). This effect wasdependent on the expression of CD40 by the tumor cells as the phenotypicchanges were absent when CD40-tumor cells were co-cultured withCD40L-modified T-cells (FIG. 23A-B). This effect was also seen in a moreclinically relevant setting in which co-cultured CD40L-modified T-cellsderived from CLL patients augmented the immunogenicity of autologous CLLcells (FIG. 24A-B). This finding demonstrates the retained ability ofT-cells to augment the immunogenicity of autologous malignant cellsthrough constitutive CD40L expression. Importantly, cell to cell contactis not a requisite to modify the tumor cell phenotype as mediacontaining elevated levels of sCD40L led to similar phenotypic changes(FIG. 28). Augmenting the immunogenicity of cancer cells through theCD40L/CD40 pathway has been shown to induce an endogenous anti-tumorresponse in previously published vaccine studies using the infusion ofautologous CLL tumor cells transduced with an adenovirus vector encodingCD40L (Ad-CD40L CLL cells).^(27,28) Infusion of tumor-specific T cellsfurther modified to constitutively express CD40L could also have asimilar capacity to induce an endogenous anti-tumor response. This mayresult in epitope spreading through the recruitment of an endogenousanti-tumor T or NK cell thereby limiting the ability of tumor escapethrough the downregulation of a single target antigen.

Dendritic cell (DC) function is impeded within the tumormicroenvironment. Normally DCs mature, migrate and present antigenwithin lymph nodes thereby stimulating the adaptive arm of the immunesystem to the presence of malignancy or pathogen.⁵ However, DC's exposedto the suppressive tumor microenvironment have a paradoxical function ofinducing T_(regs) and tolerizing tumor-specific T-cells.⁴³ To counteractthis, the CD40L/CD40 pathway can boost DCs antigen presentation,production of the pro-inflammatory cytokine IL-12, and promote CD8+T-cell cytotoxic function.^(19,20) gonist CD40 antibodies havepreviously been shown to activate DCs and boost CD8⁺ T-cell responsethereby replacing the need for CD4⁺ T-cell help.²⁶ Furthermore,CD40L-modified tumor-specific CD8⁺ T-cells have been shown to stimulatethe maturation of DCs and augment the anti-tumor responses of adoptivelytransferred CD8⁺ T-cells in tumor bearing mice.⁴⁴ To test the ability ofCD40L-modified T-cells to augment the function of human DCs, an in-vitroco-culturing experiment with autologous moDCs was used. SignificantlyCD40L-modified T-cells stimulated the secretion of IL-12p70 from moDCs(FIG. 25A-B). IL-12 is a pleiotropic cytokine with severalimmune-stimulatory functions including the ability to enhance T-cellproliferation, cytotoxic capacity, and mediate resistance to Tregsuppression as we and others have previously shown.^(7,45) The abilityof CAR/40L T-cells to stimulate IL-12 production from DCs may translateinto an improved anti-tumor effect of adoptively transferred CAR T-cellsas well as recruitment and activation of endogenous tumor specificT-cells and natural killer (NK) cells. By promoting IL-12 production inclose proximity to the tumor we anticipate minimal IL-12 relatedtoxicity in contrast to prior studies showing severe toxicity followingsystemic IL-12 administration. In addition to stimulating IL-12production, CD40L-modified T-cells promote DC maturation which in thecontext of CAR T-cell cytotoxicity should further enhance DC tumorantigen uptake and presentation resulting in recruitment/activation ofan endogenous anti-tumor response by effector T-cells and NK cells (FIG.25A-B). Taken together enhanced DC function should translate intoenhanced anti-tumor efficacy of genetically modified tumor specificT-cells through recruitment of an endogenous anti-tumor immune response.

The ability of CAR T-cells to redirect the specificity of T-cells hasbeen the demonstrated in a number of pre-clinical and clinical reports.¹We developed a retroviral vector containing the anti-CD19 CAR (1928z)and the CD40L gene (FIG. 28A). Constitutive expression of both 1928z andCD40L by T-cells is readily achievable (FIG. 28B). Significantly whentesting the cytotoxic potential of 1928z/40L T-cells against a panel ofCD19+ targets we noted increased cytotoxicity compared to T-cellsmodified with the 1928z CAR alone (FIG. 28C). Recently, Laurin andcolleagues reported enhanced cytotoxicity by CAR T-cells against tumorcell lines following CD40/IL-4 dependent upregulation of surfaceadhesion molecules which could also explain the increased cytotoxicityseen in our experiments.⁴⁶ To test the in vivo potential of CAR/CD40LT-cells a xenotransplant model using the aggressive transformedfollicular lymphoma cell line DOHH2 was used. This model has beenhistorically resistant to eradiation by 1928z T-cells (FIG. 27).However, with the added modification of CD40L our 1928z/CD40L T-cellsextend the survival of tumor bearing mice when compared to mice treatedwith 1928z T-cells alone and result in 30% long-term survival in the1928z/CD40L T-cell treated group (FIG. 27). While this modeldemonstrates a survival difference, the lack of a competent immunesystem by SCID/Beige mice makes this model unsuitable to investigate thefull benefit which constitutive CD40L expression by CAR T-cells may havein eradicating established tumors. While we observe enhanced anti-tumorefficacy in our model, this is likely related to enhanced cytotoxicityof CAR T-cells by an autocrine/paracrine CD40/CD40L pathway, and notthrough the recruitment/activation of an endogenous immune response byCD40L-modified CAR T-cells. An immune-competent syngeneic tumor modelmay be used to investigate the full effect of constitutive expression ofCD40L by CAR T cells on the tumor microenvironment and recruitment ofendogenous anti-tumor immune responses. An immune-competent syngeneicmodel of human CD19+ B-cell malignancy has recently been developed andis being utilized to assess 1928z/CD40L T-cells in the context of acompetent immune system.

The constitutive expression of CD40L on bone marrow or thymic cells hasbeen shown to result in T-lymphoproliferative disorders followinginfusion into CD40L-deficient mice.⁴⁷ The clonal populations which arosewithin the thymus following unremitting CD40L stimulation of thymocytesmay have led to malignant transformation (rather than the insertionaloncogenesis of CD40L-modified cells). While we have noted minimaltoxicity and the absence of malignant transformation following infusionof CAR/CD40L T-cells, given the concerns regarding malignant T-celltransformation, an effective suicide gene, such as iCasp9, may bedesirably included within the retroviral vector.⁴⁸

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Embodiments of the Invention

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

Some of the subject matter of this application may be related to U.S.patent application Ser. No. 12/593,751, which is the U.S. national phaseapplication, pursuant to 35 U.S.C. § 371, of International PatentApplication No.: PCT/US2008/004251, filed Mar. 8, 2010, which claims thebenefit of U.S. Provisional Application Ser. No. 60/921,144, filed Mar.30, 2007, the disclosures of which are hereby incorporated herein intheir entireties by reference.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. An immunoresponsive cell comprising: a) anantigen recognizing receptor that binds an antigen, wherein the bindingactivates the immunoresponsive cell, and b) a soluble single-chainvariable fragment (scFv) that binds a polypeptide that hasimmunosuppressive activity or immunostimulatory activity.
 2. Theimmunoresponsive cell of claim 1, wherein the soluble scFv is secreted.3. The immunoresponsive cell of claim 1, wherein the antigen recognizingreceptor and/or the soluble scFv is recombinantly expressed or expressedfrom a vector.
 4. The immunoresponsive cell of claim 1, wherein the cellis selected from the group consisting of a T cell, a Natural Killer (NK)cell, and a pluripotent stem cell from which a lymphoid cell may bedifferentiated.
 5. The immunoresponsive cell of claim 1, wherein theimmunoresponsive cell is a T cell.
 6. The immunoresponsive cell of claim5, wherein the T cell is a cytotoxic T lymphocyte (CTL) or a regulatoryT cell.
 7. The immunoresponsive cell of claim 1, wherein the antigen isa tumor antigen or a pathogen antigen.
 8. The immunoresponsive cell ofclaim 1, wherein the antigen is a tumor antigen.
 9. The immunoresponsivecell of claim 8, wherein the tumor antigen is selected from the groupconsisting of CD19, MUC16, MUC1, CAIX, CEA, CD S, CD7, CD10, CD20, CD22,CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, acytomegalovirus (CMV) infected cell antigen, EGP-2, EGP-40, EpCAM,Erb-B2, Erb-B3, Erb-B4, FBP, Fetal acetylcholine receptor, folatereceptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, K-light chain, KDR, LeY,L1 cell adhesion molecule, MAGE-A1, Mesothelin, NKG2D ligands, NY-ESO-1,oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, and WT-1.10. The immunoresponsive cell of claim 1, wherein the antigen is CD19 orMUC16.
 11. The immunoresponsive cell of claim 1, wherein theimmunosuppressive polypeptide is selected from the group consisting ofCD47, PD-1, CTLA-4, and ligands thereof.
 12. The immunoresponsive cellof claim 1, wherein the immunostimulatory polypeptide is selected fromthe group consisting of CD28, OX-40, 4-1B13, and ligands thereof. 13.The immunoresponsive cell of claim 1, wherein the antigen recognizingreceptor is a T cell receptor (TCR) or chimeric antigen receptor (CAR).14. The immunoresponsive cell of claim 1, wherein the antigenrecognizing receptor is a chimeric antigen receptor (CAR).
 15. Theimmunoresponsive cell of claim 1, wherein the CAR comprises anintracellular signaling domain.
 16. The immunoresponsive cell of claim15, wherein the intracellular signaling domain comprises a CD3-chain.17. The immunoresponsive cell of claim 15, wherein the intracellularsignaling domain comprises a signaling domain of 4-1BB, a signalingdomain of CD28 signaling domain, a signaling domain of CD97, a signalingdomain of CD11a-CD18, a signaling domain of CD2, a signaling domain ofICOS, a signaling domain of CD27, a signaling domain of CD154, asignaling domain of CDS, a signaling domain of OX40, or a combinationthereof.
 18. The immunoresponsive cell of claim 15, wherein theintracellular signaling domain comprises a signaling domain of 4-1BB ora signaling domain of CD28.
 19. A pharmaceutical composition comprisingan effective amount of an immunoresponsive cell of claim 1 and apharmaceutically acceptable excipient.
 20. A kit for treatment of atumor, a pathogen infection, or an autoimmune disorder, the kitcomprising an immunoresponsive cell of claim 1.